Site-Specific Serine Recombinases and Methods of Their Use

ABSTRACT

The present invention provides a method for obtaining site-specific recombination in a eukaryotic cell, the method comprising providing a eukaryotic cell that comprises a first recombination attachment site and a second recombination attachment site; contacting the first and second recombination attachment sites with a prokaryotic recombinase polypeptide, resulting in recombination between the recombination attachment sites, wherein the recombinase polypeptide can mediate recominbination between the first and second recombination attachment sites, the first recombination attachment site is a phage genomic recombination attachment site (attP) or a bacterial genomic recombination attachment site (attB), the second recombination site is attB or attP, and the recombinase is selected from the group consisting of a  Listeria monocytogenes  phage recombinase, a  Streptococcus pyogenes  phage recombinase, a  Bacillus subtilis  phage recombinase, a  Mycobacterium tuberculosis  phage recombinase and a  Mycobacterium smegmatis  phage recombinase, provided that when the first recombination attachment site is attB, the second recombination attachment site is attP and when the first recombination attachment site is attP, the second recombination attachment site is attB. The invention also describes compositions, vectors, and methods of use thereof, for the generation of transgenic cells, tissues, plants, and animals. The compositions, vectors and methods of the present invention are also useful in gene therapy applications.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This invention was made with government support under Grant No.70NANB1H3062, awarded by National Institute of Standards and Technology,Advance Technology Program. The Government may have certain rights inthe invention.

REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY VIA EFS-WEB

This application includes a “Sequence Listing.ST25.txt,” 37,920 bytes,created on Oct. 28, 2009, and modified on Apr. 8, 2010, and submittedelectronically via EFS-Web, which is hereby incorporated by reference inits entirety.

FIELD OF THE INVENTION

The present invention relates to the field of genetic engineering.Specifically the invention relates to compositions and methods forsite-specifically integrating, deleting, inverting, exchanging, andtranslocating a polynucleotide into the genome of a cell. The inventionalso relates to enzyme, polynucleotides, polypeptides, and vectorconstructs.

BACKGROUND OF THE INVENTION

Many bacteriophage and integrative plasmids encode site-specificrecombination systems that enable the stable incorporation of theirgenome into those of their hosts and excision of the genome from thehost genome. In these systems, the minimal requirements for therecombination reaction are a recombinase enzyme, which catalyzes therecombination event, and two recombination sites (Sadowski (1986) J.Bacteriol. 165: 341-347; Sadowski (1993) FASEB J. 7: 760-767). For phageintegration systems, these are referred to as attachment (att) sites,with an attP element from phage DNA and the attB element present in thebacterial genome. The two attachment sites can share as little sequenceidentity as a few base pairs. The recombinase protein binds to both allsites and catalyzes a conservative and reciprocal exchange of DNAstrands that result in integration of the circular phage or plasmid DNAinto host DNA. Additional phage or host factors, such as the DNA bendingprotein IHF, integration host factor, may be required for an efficientreaction (Friedman (1988) Cell 55:545-554; Finkel & Johnson (1992) Mol.Microbiol. 6: 3257-3265). Phage integrases, in association with otherhost and/or phage factors, also excise the phage genome from thebacterial genome during the lytic phase of bacteriophages growth cycle.Several methods have been developed allowing the manipulation ofmammalian genomes in order to elucidate the relevance and function ofparticular genes of interest. Among them, the development of transgenicmouse strains and gene-targeting technologies have turned out to beparticularly useful (Brandon, E. P., Idzerda, R. L. and McKnight, G. S.(1995) Curr Biol, 5, 625-34; Brandon, E. P., Idzerda, R. L. andMcKnight, G. S. (1995) Curr Biol, 5, 758-65). These techniques haveundergone a new advance with the characterization and application ofsite-specific recombinases (Kilby, N. J., Snaith, M. R. and Murray, J.A. (1993) Trends Genet, 9,413-21).

Site-specific recombinases can be separated into two major families. Thefirst one (the Int family or tyrosine recombinase family) comprisesthose enzymes that catalyze recombination between sites located eitherin the same DNA molecule (intramolecular recombination leading toresolution, excision, or inversion) or in separate DNA molecules(intermolecular recombination leading to integration) (Sauer, B. (1993)Methods Enzymol, 225, 890-900; Dymecki, S. M. (1996) Proc Natl Acad SciUSA 93, 6191-6; Abremski, K. and Hoess, R. (1984) J Biol Chem, 259,1509-14; Nash, H. A. (1996) in Escherichia coli and Salmonella cellularand molecular biology., ed. F. C. Neidhart, R. I. Curtis, J. L.Ingraham, E. C. C. Lin, K. B. Low, B. Magasanik, W. S. Rezaikoff, M.Riley, M. Schaechter and H. E. Umbager (A.S.M. Press, Washington D.C.),pp. 2363-7). The latter property has been exploited to allow targetedinsertion of specific sequences in precise locations (Sauer, B. andHenderson, N. (1990) The New Biologist, 2, 441-9; Fukushige, S. andSauer, B. (1992) Proc. Natl. Acad. Sci. USA, 89, 7905-9). Therecombinases that have been used for manipulating mammalian genomes havebeen mainly the Cre and the Flp proteins, which belong to the Int family(Kilby, N. J., Snaith, M. R. and Murray, J. A. (1993) Trends Genet,9,413-21). The target sequences for these enzymes, named loxP sites forthe Cre enzyme and FRT for the Flp enzyme, consist of a short invertedrepeat to which the protein binds. The recombination process isoperative through long distances (up to 70 kb) in the genome. Usingthese enzymes, several authors have reported site- and tissue-specificDNA recombination in murine models (DiSanto, J. P., Muller, W., Guy, G.D., Fischer, A. and Rajewsky, K. (1995) Proc Natl Acad Sci USA, 92,377-81; Gu, H., Marth, J. D., Orban, P. C., Massmann, H. and Rajewsky,K. (1994) Science, 265, 103-6; Kuhn, R., Schwenk, F., Aguet, M. andRajewsky, K. (1995) Science. 269, 1427-9; Orban, P. C., Chui, D. andMarth, J. D. (1992) Proc. Natl. Acad. Sci. USA, 89, 6861-5), chromosomaltranslocations in plants and animals (Deursen, J. v., Fornerod, M.,Rees, B. v. and Grosveld, G. (1995) Proc. Natl. Acad. Sci. USA. 92,7376-80; Medberry, S. L., Dale, E., Qin, M. and Ow, D. W. (1995) NucleicAcids Res, 23, 485-90; Osborne, B. I., Wirtz, U. and Baker, B. (1995)Plant J, 7, 687-701) and targeted induction of specific genes (Pichel,J. G., Lakso, M. and Westphal, H. (1993) Oncogene, 8, 3333-42). TheCre-loxP system has also been used in combination with induciblepromoters, such as the interferon gamma inducible promoter, that wasused to provoke gene ablation in liver with high efficiency and to aless extent in other tissues (Kuhn, R., Schwenk, F., Aguet, M. andRajewsky, K. (1995) Science, 269, 1427-9). This site-specificrecombination system, however, only allows the induction of a reducednumber of recombination events in the same genome. Since eachrecombination reaction leaves a target sequence for the recombinase inthe genome at the crossover site, and because recombinases (e.g. Cre andFlp) can catalyze intermolecular recombination, the whole process maylead to undesired chromosomal rearrangements.

The second family of recombinases are collectively termedresolvases/invertases family or serine family (Grindley, N. D. F. (1994)in Nucleic Acids and Molecular Biology, ed. F. Eckstein and D. M. J.Lilley (Springer-Verlag, Berlin), pp. 236-67, (Smith, M. C. and Thorpe,H. M. (2000) Mol. Microbiol., 44, 299-307)). These site-specificrecombinases, which include enzymes that catalyze intramolecular andintermolecular reactions, could have an advantage over the Int family ofrecombinases. Serine recombinases that catalyze phage integration(integrases) are especially well adapted for use as genetic engineeringtools. So far three serine recombinases, φC31, R4 and TP901-1, have beenexamined in mammalian cells (Groth, A. C. and Calos, M. P. (2004) J.Mol. Biol. 335, 667-678). These recombinases were observed to beautonomous, to have simple att sequences and have the ability tofunction in mammalian cells. As little or no recombination between anycombination of sites other than attP or attB has been observed, theintegrations are unidirectional and there is a high integrationfrequency. Serine recombinases provide a significant advantage over theprior recombination systems employing the use of members of the Intfamily of recombinases. These enzymes have numerous applications. Oneway is the placement of all sites into the genome of an organism and useas targets for recombination.

Applicant has identified novel serine recombinases that demonstraterobust activity in various mammalian cells and in plant cells, as wellas the ability to stably integrate a polynucleotide into the genome of ahost cell or excise a polynucleotide from the genome of a host cell.

SUMMARY OF THE INVENTION

The present invention provides compositions and methods for obtainingstable, site-specific recombination in a eukaryotic cell. Contrary topreviously described methods for site-specific recombination, thepresent recombinases and methods of their use provide for stable,irreversible, site-specific recombination.

The compositions of the present invention provide for a recombinasepolypeptide that mediates site-specific recombination between a firstrecombination site and a second recombination site. In some embodiments,the nucleic acids further include recombination sites recognized by therecombinase polypeptide.

The methods involve providing a eukaryotic cell that comprises a firstrecombination site and a second recombination site, which secondrecombination site can serve as a substrate for recombination with thefirst recombination site. The first and the second recombination sitesare contacted with a prokaryotic recombinase polypeptide, resulting inrecombination between the recombination sites. Either or both of therecombination sites can be present in a chromosome of the eukaryoticcell. In some embodiments, one of the recombination sites is present inthe chromosome and the other is included within a nucleic acid that isto be integrated into the chromosome.

The invention also provides eukaryotic cells that contain a prokaryoticrecombinase polypeptide or a nucleic acid that encodes a prokaryoticrecombinase. In these embodiments, the recombinase can mediatesite-specific recombination between a first recombination site and asecond recombination site that can serve as a substrate forrecombination with the first recombination site. In preferredembodiments the recombinases are selected from the group consisting of aListeria monocytogenes phage, a Streptococcus pyogenes phage, a Bacillussubtilis phage, a Mycobacterium tuberculosis phage and a Mycobacteriumsmegmatis phage. More preferably, the recombinase is selected from thegroup consisting of A118 recombinase, SF370.1 recombinase, SPβc2recombinase, φRv1 recombinase, and Bxb1 recombinase.

In additional embodiments, the invention provides methods for obtaininga eukaryotic cell having a stably integrated polynucleotide sequence.These methods involve introducing a nucleic acid into a eukaryotic cellthat comprises a first recombination site, wherein the nucleic acidcomprises the transgene of interest and a second recombination sitewhich can serve as a substrate for recombination with the firstrecombination site. The first and second recombination sites arecontacted with a prokaryotic recombinase polypeptide. The recombinasepolypeptide catalyzes recombination between the first and secondrecombination sites, resulting in integration of the nucleic acid at thefirst recombination site.

The ability of phage recombinases to specifically and efficiently directrecombination between DNA sequences in living cells makes thempotentially useful in a variety of genetic engineering applications.Such applications include integration, excision, inversion,translocation and cassette exchange of polynucleotide sequences.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts a schematic representation of the TransientIntramolecular Recombination Assay (TIRA) used to assay the ability ofthe recombinase to detect recombination between attP and attB sites on atarget or assay plasmid as described in the Examples.

FIG. 2 demonstrates the results of the TIRA for various recombinasesperformed in human embryonic kidney (HEK293) cells.

FIG. 3 demonstrates the results of the TIRA for various recombinasesperformed in mouse NIH3T3 cells.

FIG. 4 demonstrates the results of the TIRA for various recombinasesperformed in Chinese hamster ovary (CHO) cells.

FIG. 5 demonstrates the results of the TIRA for various recombinasesperformed in human HeLa cells.

FIG. 6 demonstrates the results of the TIRA for various recombinasesperformed in rat bone marrow stromal cells.

FIG. 7 demonstrates the results of the TIRA for various recombinasesperformed in mouse neural stem cells.

FIG. 8 demonstrates the results of the TIRA assay for A118 recombinaseperformed in tobacco BY2 cells.

FIG. 9 depicts a schematic representation of stable integration ofplasmid DNA containing attP or attB sequence into HEK293 chromosomecontaining the attB or attP site.

FIG. 10 demonstrates the results of PCR amplification of attL and attRsites following stable integration of plasmid DNA containing attP orattB sequence into HEK293 cell chromosome containing the attB or attPsite.

FIG. 11 depicts a schematic representation of excision of stablyintegrated STOP sequence and activation of luciferase activity due torecombinase.

FIG. 12 demonstrates the results of excision of stably integrated STOPsequence and activation of luciferase activity due to recombinase.

FIG. 13 depicts a schematic representation of insertion or integrationof a plasmid containing attP or attB recombination site at the nativepseudo attB or pseudo attP site present in HEK293 cells.

FIG. 14 demonstrates the nucleotide sequences of native pseudo attBsites for SF370.1 and SPβcp recombinases identified in HEK293 cells.

DESCRIPTION OF THE PREFERRED EMBODIMENT Definitions

In this disclosure, a number of terms and abbreviations are used. Thefollowing definitions are provided and should be helpful inunderstanding the scope and practice of the present invention.

In a specific embodiment, the term “about” or “approximately” meanswithin 20%, preferably within 10%, more preferably within 5%, and evenmore preferably within 1% of a given value or range.

“Recombinase” as used herein refers to a group of enzymes that canfacilitate site-specific recombination between defined sites, where thesites are physically separated on a single DNA molecule or where thesites reside on separate DNA molecules. The DNA sequences of the definedrecombination sites are not necessarily identical. Initiation ofrecombination depends on protein-DNA interaction, within the group thereare large number of proteins that catalyze phage integration andexcision (e.g., λ integrase, φC31), resolution of circular plasmids(e.g., Tn3, gamma delta, Cre, Flp), DNA inversion for expression ofalternate genes (e.g., Hin, Gin, Pin), assembly of genes duringdevelopment (e.g., Anabaena nitrogen fixation genes), and transposition(e.g., IS607 transposon). Most site-specific recombinases fall into oneof the two families, based on evolutionary and mechanistic relatedness.These are λ integrase family or tyrosine recombinases (e.g., Cre, Flp,Xer D) and resolvase/integrase family or serine recombinase family(e.g., φC31, TP901-1, Tn3, gamma delta).

“Recombination attachment sites” are specific polynucleotide sequencesthat are recognized by the recombinase enzymes described herein.Typically, two different sites are involved (termed “complementarysites”), one present in the target nucleic acid (e.g., a chromosome orepisome of a eukaryote or prokaryote) and another on the nucleic acidthat is to be integrated at the target recombination site. The terms“attB” and “attP,” which refer to attachment (or recombination) sitesoriginally from a bacterial target and a phage donor, respectively, areused herein although recombination sites for particular enzymes may havedifferent names. The recombination sites typically include left andright arms separated by a core or spacer region. Thus, an attBrecombination site consists of BOB′, where B and B′ are the left andright arms, respectively, and O is the core region. Similarly, attP isPOP′, where P and P′ are the arms and O is again the core region. Uponrecombination between the attB and attP sites, and concomitantintegration of a nucleic acid at the target, the recombination sitesthat flank the integrated DNA are referred to as “attL” and “attR.” TheattL and attR sites, using the terminology above, thus consist of BOP′and POB′, respectively. In some representations herein, the “O” isomitted and attB and attP, for example, are designated as BB′ and PP′,respectively.

The term “substantially free” means that a composition comprising “A”(where “A” is a single protein, DNA molecule, vector, recombinant hostcell, etc.) is substantially free of “B” (where “B” comprises one ormore contaminating proteins, DNA molecules, vectors, etc.) when at leastabout 75% by weight of the proteins, DNA, vectors (depending on thecategory of species to which A and B belong) in the composition is “A”.Preferably, “A” comprises at least about 90% by weight of the A+Bspecies in the composition, most preferably at least about 99% byweight. It is also preferred that a composition, which is substantiallyfree of contamination, contain only a single molecular weight specieshaving the activity or characteristic of the species of interest.

The term “isolated” for the purposes of the present invention designatesa biological material (nucleic acid or protein) that has been removedfrom its original environment (the environment in which it is naturallypresent). For example, a polynucleotide present in the natural state ina plant or an animal is not isolated, however the same polynucleotideseparated from the adjacent nucleic acids in which it is naturallypresent, is considered “isolated”. The term “purified” does not requirethe material to be present in a form exhibiting absolute purity,exclusive of the presence of other compounds. It is rather a relativedefinition.

A polynucleotide is in the “purified” state after purification of thestarting material or of the natural material by at least one order ofmagnitude, preferably 2 or 3 and preferably 4 or 5 orders of magnitude.

A “nucleic acid” is a polymeric compound comprised of covalently linkedsubunits called nucleotides. Nucleic acid includes polyribonucleic acid(RNA) and polydeoxyribonucleic acid (DNA), both of which may besingle-stranded or double-stranded.

DNA includes but is not limited to cDNA, genomic DNA, plasmids DNA,synthetic DNA, and semi-synthetic DNA. DNA may be linear, circular, orsupercoiled.

A “nucleic acid molecule” refers to the phosphate ester polymeric formof ribonucleosides (adenosine, guanosine, uridine or cytidine; “RNAmolecules”) or deoxyribonucleosides (deoxyadenosine, deoxyguanosine,deoxythymidine, or deoxycytidine; “DNA molecules”), or any phosphoesteranalogs thereof, such as phosphorothioates and thioesters, in eithersingle stranded form, or a double-stranded helix. Double strandedDNA-DNA, DNA-RNA and RNA-RNA helices are possible. The term nucleic acidmolecule, and in particular DNA or RNA molecule, refers only to theprimary and secondary structure of the molecule, and does not limit itto any particular tertiary forms. Thus, this term includesdouble-stranded DNA found, inter alia, in linear or circular DNAmolecules (e.g., restriction fragments), plasmids, and chromosomes. Indiscussing the structure of particular double-stranded DNA molecules,sequences may be described herein according to the normal convention ofgiving only the sequence in the 5′ to 3′ direction along thenon-transcribed strand of DNA (i.e., the strand having a sequencehomologous to the mRNA). A “recombinant DNA molecule” is a DNA moleculethat has undergone a molecular biological manipulation.

The term “fragment” will be understood to mean a nucleotide sequence ofreduced length relative to the reference nucleic acid and comprising,over the common portion, a nucleotide sequence identical to thereference nucleic acid. Such a nucleic acid fragment according to theinvention may be, where appropriate, included in a larger polynucleotideof which it is a constituent. Such fragments comprise, or alternativelyconsist of, oligonucleotides ranging in length from at least 6, 8, 9,10, 12, 15, 18, 20, 21, 22, 23, 24, 25, 30, 39, 40, 42, 45, 48, 50, 51,54, 57, 60, 63, 66, 70, 75, 78, 80, 90, 100, 105, 120, 135, 150, 200,300, 500, 720, 900, 1000 or 1500 consecutive nucleotides of a nucleicacid according to the invention.

As used herein, an “isolated nucleic acid fragment” is a polymer of RNAor DNA that is single- or double-stranded, optionally containingsynthetic, non-natural or altered nucleotide bases. An isolated nucleicacid fragment in the form of a polymer of DNA may be comprised of one ormore segments of cDNA, genomic DNA or synthetic DNA.

A “gene” refers to an assembly of nucleotides that encode a polypeptide,and includes cDNA and genomic DNA nucleic acids. “Gene” also refers to anucleic acid fragment that expresses a specific protein or polypeptide,including regulatory sequences preceding (5′ non-coding sequences) andfollowing (3′ non-coding sequences) the coding sequence. “Native gene”refers to a gene as found in nature with its own regulatory sequences.“Chimeric gene” refers to any gene that is not a native gene, comprisingregulatory and/or coding sequences that are not found together innature. Accordingly, a chimeric gene may comprise regulatory sequencesand coding sequences that are derived from different sources, orregulatory sequences and coding sequences derived from the same source,but arranged in a manner different than that found in nature. A chimericgene may comprise coding sequences derived from different sources and/orregulatory sequences derived from different sources. “Endogenous gene”refers to a native gene in its natural location in the genome of anorganism. A “foreign” gene or “heterologous” gene refers to a gene notnormally found in the host organism, but that is introduced into thehost organism by gene transfer. Foreign genes can comprise native genesinserted into a non-native organism, or chimeric genes. A “transgene” isa gene that has been introduced into the genome by a transformationprocedure.

“Heterologous” DNA refers to DNA not naturally located in the cell, orin a chromosomal site of the cell. Preferably, the heterologous DNAincludes a gene foreign to the cell.

The term “genome” includes chromosomal as well as mitochondrial,chloroplast and viral DNA or RNA.

A nucleic acid molecule is “hybridizable” to another nucleic acidmolecule, such as a cDNA, genomic DNA, or RNA, when a single strandedform of the nucleic acid molecule can anneal to the other nucleic acidmolecule under the appropriate conditions of temperature and solutionionic strength (see Sambrook et al., 1989 intfra). Hybridization andwashing conditions are well known and exemplified in Sambrook, J.,Fritsch, E. F. and Maniatis, T. Molecular Cloning: A Laboratory Manual,Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor(1989), particularly Chapter 11 and Table 11.1 therein (entirelyincorporated herein by reference). The conditions of temperature andionic strength determine the “stringency” of the hybridization.

Stringency conditions can be adjusted to screen for moderately similarfragments, such as homologous sequences from distantly relatedorganisms, to highly similar fragments, such as genes that duplicatefunctional enzymes from closely related organisms. For preliminaryscreening for homologous nucleic acids, low stringency hybridizationconditions, corresponding to a T_(m) of 55°, can be used, e.g., 5×SSC,0.1% SDS, 0.25% milk, and no formamide; or 30% formamide, 5×SSC, 0.5%SDS). Moderate stringency hybridization conditions correspond to ahigher T., e.g., 40% formamide, with 5× or 6×SCC. High stringencyhybridization conditions correspond to the highest T., e.g., 50%formamide, 5× or 6×SCC.

Hybridization requires that the two nucleic acids contain complementarysequences, although depending on the stringency of the hybridization,mismatches between bases are possible. The term “complementary” is usedto describe the relationship between nucleotide bases that are capableof hybridizing to one another. For example, with respect to DNA,adenosine is complementary to thymine and cytosine is complementary toguanine. Accordingly, the instant invention also includes isolatednucleic acid fragments that are complementary to the complete sequencesas disclosed or used herein as well as those substantially similarnucleic acid sequences.

In a specific embodiment of the invention, polynucleotides are detectedby employing hybridization conditions comprising a hybridization step atT_(m) of 55° C., and utilizing conditions as set forth above. In apreferred embodiment, the Tm is 60° C.; in a more preferred embodiment,the T_(m) is 63° C.; in an even more preferred embodiment, the T_(m) is65° C.

Post-hybridization washes also determine stringency conditions. One setof preferred conditions uses a series of washes starting with 6×SSC,0.5% SDS at room temperature for 15 minutes (min), then repeated with2×SSC, 0.5% SDS at 45′C for 30 minutes, and then repeated twice with0.2×SSC, 0.5% SDS at 50° C. for 30 minutes. A more preferred set ofstringent conditions uses higher temperatures in which the washes areidentical to those above except for the temperature of the final two 30min washes in 0.2×SSC, 0.5% SDS was increased to 60° C. Anotherpreferred set of highly stringent conditions uses two final washes in0.1×SSC, 0.1% SDS at 65° C. Hybridization requires that the two nucleicacids comprise complementary sequences, although depending on thestringency of the hybridization, mismatches between bases are possible.

The appropriate stringency for hybridizing nucleic acids depends on thelength of the nucleic acids and the degree of complementation, variableswell known in the art. The greater the degree of similarity or homologybetween two nucleotide sequences, the greater the value of T_(m) forhybrids of nucleic acids having those sequences. The relative stability(corresponding to higher T_(m)) of nucleic acid hybridizations decreasesin the following order: RNA:RNA, DNA:RNA, DNA:DNA. For hybrids ofgreater than 100 nucleotides in length, equations for calculating Tmhave been derived (see Sambrook et al., supra. 9.50-0.51). Forhybridization with shorter nucleic acids, i.e., oligonucleotides, theposition of mismatches becomes more important, and the length of theoligonucleotide determines its specificity (see Sambrook et al., supra,11.7-11.8).

In a specific embodiment of the invention, polynucleotides are detectedby employing hybridization conditions comprising a hybridization step inless than 500 mM salt and at least 37 degrees Celsius, and a washingstep in 2×SSPE at at least 63-degrees Celsius. In a preferredembodiment, the hybridization conditions comprise less than 200 mM saltand at least 37 degrees Celsius for the hybridization step. In a morepreferred embodiment, the hybridization conditions comprise 2×SSPE and63 degrees Celsius for both the hybridization and washing steps.

In one embodiment, the length for a hybridizable nucleic acid is atleast about 10 nucleotides. Preferably a minimum length for ahybridizable nucleic acid is at least about 15 nucleotides; morepreferably at least about 20 nucleotides; and most preferably the lengthis at least 30 nucleotides. Furthermore, the skilled artisan willrecognize that the temperature and wash solution salt concentration maybe adjusted as necessary according to factors such as length of theprobe.

The term “probe” refers to a single-stranded nucleic acid molecule thatcan base pair with a complementary single stranded target nucleic acidto form a double-stranded molecule.

As used herein, the term “oligonucleotide” refers to a nucleic acid,generally of at least 18 nucleotides, that is hybridizable to a genomicDNA molecule, a cDNA molecule, a plasmid DNA or an mRNA molecule.Oligonucleotides can be labeled, e.g., with ³²P-nucleotides ornucleotides to which a label, such as biotin, has been covalentlyconjugated. A labeled oligonucleotide can be used as a probe to detectthe presence of a nucleic acid. Oligonucleotides (one or both of whichmay be labeled) can be used as PCR primers, either for cloning fulllength or a fragment of a nucleic acid, or to detect the presence of anucleic acid. An oligonucleotide can also be used to form a triple helixwith a DNA molecule. Generally, oligonucleotides are preparedsynthetically, preferably on a nucleic acid synthesizer. Accordingly,oligonucleotides can be prepared with non-naturally occurringphosphoester analog bonds, such as thioester bonds, etc.

A “primer” is an oligonucleotide that hybridizes to a target nucleicacid sequence to create a double stranded nucleic acid region that canserve as an initiation point for DNA synthesis under suitableconditions. Such primers may be used in a polymerase chain reaction.

“Polymerase chain reaction” is abbreviated PCR and means an in vitromethod for enzymatically amplifying specific nucleic acid sequences. PCRinvolves a repetitive series of temperature cycles with each cyclecomprising three stages: denaturation of the template nucleic acid toseparate the strands of the target molecule, annealing a single strandedPCR oligonucleotide primer to the template nucleic acid, and extensionof the annealed primer(s) by DNA polymerase. PCR provides a means todetect the presence of the target molecule and, under quantitative orsemi-quantitative conditions, to determine the relative amount of thattarget molecule within the starting pool of nucleic acids.

“Reverse transcription-polymerase chain reaction” is abbreviated RT-PCRand means an in vitro method for enzymatically producing a target cDNAmolecule or molecules from an RNA molecule or molecules, followed byenzymatic amplification of a specific nucleic acid sequence or sequenceswithin the target cDNA molecule or molecules as described above. RT-PCRalso provides a means to detect the presence of the target molecule and,under quantitative or semi-quantitative conditions, to determine therelative amount of that target molecule within the starting pool ofnucleic acids.

A DNA “coding sequence” is a double-stranded DNA sequence that istranscribed and translated into a polypeptide in a cell in vitro or invivo when placed under the control of appropriate regulatory sequences.“Suitable regulatory sequences” refer to nucleotide sequences locatedupstream (5′ non-coding sequences), within, or downstream (3′ non-codingsequences) of a coding sequence, and which influence the transcription,RNA processing or stability, or translation of the associated codingsequence. Regulatory sequences may include promoters, translation leadersequences, introns, polyadenylation recognition sequences, RNAprocessing site, effector binding site and stem-loop structure. Theboundaries of the coding sequence are determined by a start codon at the5′ (amino) terminus and a translation stop codon at the 3′ (carboxyl)terminus. A coding sequence can include, but is not limited to,prokaryotic sequences, cDNA from mRNA, genomic DNA sequences, and evensynthetic DNA sequences. If the coding sequence is intended forexpression in a eukaryotic cell, a polyadenylation signal andtranscription termination sequence will usually be located 3′ to thecoding sequence.

“Open reading frame” is abbreviated ORF and means a length of nucleicacid sequence. either DNA, cDNA or RNA, that comprises a translationstart signal or initiation codon, such as an ATG or AUG, and atermination codon and can be potentially translated into a polypeptidesequence.

The term “head-to-head” is used herein to describe the orientation oftwo polynucleotide sequences in relation to each other. Twopolynucleotides are positioned in a head-to-head orientation when the 5′end of the coding strand of one polynucleotide is adjacent to the 5′ endof the coding strand of the other polynucleotide, whereby the directionof transcription of each polynucleotide proceeds away from the 5′ end ofthe other polynucleotide. The term “head-to-head” may be abbreviated(5′)-to-(5′) and may also be indicated by the symbols (←→) or(3′←5′5′→3′).

The term “tail-to-tail” is used herein to describe the orientation oftwo polynucleotide sequences in relation to each other. Twopolynucleotides are positioned in a tail-to-tail orientation when the 3′end of the coding strand of one polynucleotide is adjacent to the 3′ endof the coding strand of the other polynucleotide, whereby the directionof transcription of each polynucleotide proceeds toward the otherpolynucleotide. The term “tail-to-tail” may be abbreviated (3′)-to-(3′)and may also be indicated by the symbols (←→) or (5′→3′3′←5′).

The term “head-to-tail” is used herein to describe the orientation oftwo polynucleotide sequences in relation to each other. Twopolynucleotides are positioned in a head-to-tail orientation when the 5′end of the coding strand of one polynucleotide is adjacent to the 3′ endof the coding strand of the other polynucleotide, whereby the directionof transcription of each polynucleotide proceeds in the same directionas that of the other polynucleotide. The term “head-to-tail” may beabbreviated (5′)-to-(3′) and may also be indicated by the symbols (→→)or (5′→3′5′→3′).

The term “downstream” refers to a nucleotide sequence that is located 3′to reference nucleotide sequence. In particular, downstream nucleotidesequences generally relate to sequences that follow the starting pointof transcription. For example, the translation initiation codon of agene is located downstream of the start site of transcription.

The term “upstream” refers to a nucleotide sequence that is located 5′to reference nucleotide sequence. In particular, upstream nucleotidesequences generally relate to sequences that are located on the 5′ sideof a coding sequence or starting point of transcription. For example,most promoters are located upstream of the start site of transcription.

The terms “restriction endonuclease” and “restriction enzyme” refer toan enzyme that binds and cuts within a specific nucleotide sequencewithin double stranded DNA.

“Homologous recombination” refers to the insertion of a foreign DNAsequence into another DNA molecule, e.g., insertion of a vector in achromosome. Preferably, the vector targets a specific chromosomal sitefor homologous recombination. For specific homologous recombination, thevector will contain sufficiently long regions of homology to sequencesof the chromosome to allow complementary binding and incorporation ofthe vector into the chromosome. Longer regions of homology, and greaterdegrees of sequence similarity, may increase the efficiency ofhomologous recombination.

Several methods known in the art may be used to propagate apolynucleotide according to the invention. Once a suitable host systemand growth conditions are established, recombinant expression vectorscan be propagated and prepared in quantity. As described herein, theexpression vectors which can be used include, but are not limited to,the following vectors or their derivatives: human or animal viruses suchas vaccinia virus or adenovirus; insect viruses such as baculovirus;yeast vectors; bacteriophage vectors (e.g., lambda), and plasmid andcosmid DNA vectors, to name but a few.

A “vector” is any means for the cloning of and/or transfer of a nucleicacid into a host cell. A vector may be a replicon to which another DNAsegment may be attached so as to bring about the replication of theattached segment. A “replicon” is any genetic element (e.g., plasmid,phage, cosmid, chromosome, virus) that functions as an autonomous unitof DNA replication in vivo, i.e., capable of replication under its owncontrol. The term “vector” includes both viral and non viral means forintroducing the nucleic acid into a cell in vitro, ex viva or in vivo. Alarge number of vectors known in the art may be used to manipulatenucleic acids, incorporate response elements and promoters into genes,etc. Possible vectors include, for example, plasmids or modified virusesincluding, for example bacteriophages such as lambda derivatives, orplasmids such as pBR322 or pUC plasmid derivatives, or the Bluescriptvector. For example, the insertion of the DNA fragments corresponding toresponse elements and promoters into a suitable vector can beaccomplished by ligating the appropriate DNA fragments into a chosenvector that has complementary cohesive termini. Alternatively, the endsof the DNA molecules may be enzymatically modified or any site may beproduced by ligating nucleotide sequences (linkers) into the DNAtermini. Such vectors may be engineered to contain selectable markergenes that provide for the selection of cells that have incorporated themarker into the cellular genome. Such markers allow identificationand/or selection of host cells that incorporate and express the proteinsencoded by the marker.

Viral vectors, and particularly retroviral vectors, have been used in awide variety of gene delivery applications in cells, as well as livinganimal subjects. Viral vectors that can be used include but are notlimited to retrovirus, adeno-associated virus, pox, baculovirus,vaccinia, herpes simplex, Epstein-Barr, adenovirus, geminivirus, andcaulimovirus vectors. Non-viral vectors include plasmids, liposomes,electrically charged lipids (cytofectins), DNA-protein complexes, andbiopolymers. In addition to a nucleic acid, a vector may also compriseone or more regulatory regions, and/or selectable markers useful inselecting, measuring, and monitoring nucleic acid transfer results(transfer to which tissues, duration of expression, etc.).

The term “plasmid” refers to an extra chromosomal element often carryinga gene that is not part of the central metabolism of the cell, andusually in the form of circular double-stranded DNA molecules. Suchelements may be autonomously replicating sequences, genome integratingsequences, phage or nucleotide sequences, linear, circular, orsupercoiled, of a single- or double-stranded DNA or RNA, derived fromany source, in which a number of nucleotide sequences have been joinedor recombined into a unique construction which is capable of introducinga promoter fragment and DNA sequence for a selected gene product alongwith appropriate 3′ untranslated sequence into a cell.

A “cloning vector” is a “replicon”, which is a unit length of a nucleicacid, preferably DNA, that replicates sequentially and which comprisesan origin of replication, such as a plasmid, phage or cosmid, to whichanother nucleic acid segment may be attached so as to bring about thereplication of the attached segment. Cloning vectors may be capable ofreplication in one cell type and expression in another (“shuttlevector”).

Vectors may be introduced into the desired host cells by methods knownin the art, e.g., transfection, electroporation, microinjection,transduction, cell fusion, DEAE dextran, calcium phosphateprecipitation, lipofection (lysosome fusion), use of a gene gun, or aDNA vector transporter (see, e.g., Wu et al., 1992, J. Biol. Chem. 267:963-967; Wu and Wu, 1988, J. Biol. Chem. 263: 14621-14624; and Hartmutet al., Canadian Patent Application No. 2,012,311, filed Mar. 15, 1990).

A polynucleotide according to the invention can also be introduced invivo by lipofection. For the past decade, there has been increasing useof liposomes for encapsulation and transfection of nucleic acids invitro. Synthetic cationic lipids designed to limit the difficulties anddangers encountered with liposome-mediated transfection can be used toprepare liposomes for in vivo transfection of a gene encoding a marker(Feigner et al., 1987, Proc. Natl. Acad. Sci. U.S.A. 84: 7413; Mackey,et al., 1988, Proc. Natl. Acad. Sci. U.S.A. 85:8027-8031; and Ulmer etal., 1993, Science 259: 1745-1748). The use of cationic lipids maypromote encapsulation of negatively charged nucleic acids, and alsopromote fusion with negatively charged cell membranes (Feigner andRingold, 1989, Science 337:387-388). Particularly useful lipid compoundsand compositions for transfer of nucleic acids are described inInternational Patent Publications WO95/18863 and WO96/17823, and in U.S.Pat. No. 5,459,127. The use of lipofection to introduce exogenous genesinto the specific organs in vi w has certain practical advantages.Molecular targeting of liposomes to specific cells represents one areaof benefit. It is clear that directing transfection to particular celltypes would be particularly preferred in a tissue with cellularheterogeneity, such as pancreas, liver, kidney, and the brain. Lipidsmay be chemically coupled to other molecules for the purpose oftargeting (Mackey, et al., 1988, supra). Targeted peptides, e.g.,hormones or neurotransmitters, and proteins such as antibodies, ornon-peptide molecules could be coupled to liposomes chemically.

Other molecules are also useful for facilitating transfection of anucleic acid in vivo. such as a cationic oligopeptide (e.g.,WO95/21931), peptides derived from DNA binding proteins (e.g.,WO96/25508), or a cationic polymer (e.g., WO95/21931).

It is also possible to introduce a vector in vivo as a naked DNA plasmid(see U.S. Pat. Nos. 5,693,622, 5,589,466 and 5,580,859).Receptor-mediated DNA delivery approaches can also be used (Curiel etal., 1992, Hum. Gene Ther. 3: 147-154; and Wu and Wu, 1987, J. Biol.Chem. 262: 4429-4432).

The term “transfection” means the uptake of exogenous or heterologousRNA or DNA by a cell. A cell has been “transfected” by exogenous orheterlogous RNA or DNA when such RNA or DNA has been introduced insidethe cell. A cell has been “transformed” by exogenous or heterologous RNAor DNA when the transfected RNA or DNA effects a phenotypic change. Thetransforming RNA or DNA can be integrated (covalently linked) intochromosomal DNA making up the genome of the cell.

“Transformation” refers to the transfer of a nucleic acid fragment intothe genome of a host organism, resulting in genetically stableinheritance. Host organisms containing the transformed nucleic acidfragments are referred to as “transgenic” or “recombinant” or“transformed” organisms.

The term “genetic region” will refer to a region of a nucleic acidmolecule or a nucleotide sequence that comprises a gene encoding apolypeptide.

In addition, the recombinant vector comprising a polynucleotideaccording to the invention may include one or more origins forreplication in the cellular hosts in which their amplification or theirexpression is sought, markers or selectable markers.

The term “selectable marker” means an identifying factor, usually anantibiotic or chemical resistance gene, that is able to be selected forbased upon the marker gene's effect, i.e., resistance to an antibiotic,resistance to a herbicide, colorimetric markers, enzymes, fluorescentmarkers, and the like, wherein the effect is used to track theinheritance of a nucleic acid of interest and/or to identify a cell ororganism that has inherited the nucleic acid of interest. Examples ofselectable marker genes known and used in the art include: genesproviding resistance to ampicillin, streptomycin, gentamycin, kanamycin,hygromycin, bialaphos herbicide, sulfonamide, and the like; and genesthat are used as phenotypic markers, i.e., anthocyanin regulatory genes,isopentanyl transferase gene, and the like.

The term “reporter gene” means a nucleic acid encoding an identifyingfactor that is able to be identified based upon the reporter gene'seffect, wherein the effect is used to track the inheritance of a nucleicacid of interest, to identify a cell or organism that has inherited thenucleic acid of interest, and/or to measure gene expression induction ortranscription. Examples of reporter genes known and used in the artinclude: luciferase (Luc), green fluorescent protein (GFP),chloramphenicol acetyltransferase (CAT), β-galactosidase (LacZ),β-glucuronidase (Gus), and the like. Selectable marker genes may also beconsidered reporter genes.

“Promoter” refers to a DNA sequence capable of controlling theexpression of a coding sequence or functional RNA. In general, a codingsequence is located 3′ to a promoter sequence. Promoters may be derivedin their entirety from a native gene, or be composed of differentelements derived from different promoters found in nature, or evencomprise synthetic DNA segments. It is understood by those skilled inthe art that different promoters may direct the expression of a gene indifferent tissues or cell types, or at different stages of development,or in response to different environmental or physiological conditions.Promoters that cause a gene to be expressed in most cell types at mosttimes are commonly referred to as “constitutive promoters”. Promotersthat cause a gene to be expressed in a specific cell type are commonlyreferred to as “cell-specific promoters” or “tissue-specific promoters”.Promoters that cause a gene to be expressed at a specific stage ofdevelopment or cell differentiation are commonly referred to as“developmentally-specific promoters” or “cell differentiation-specificpromoters”. Promoters that are induced and cause a gene to be expressedfollowing exposure or treatment of the cell with an agent, biologicalmolecule, chemical, ligand, light, or the like that induces the promoterare commonly referred to as “inducible promoters” or “regulatablepromoters”. It is further recognized that since in most cases the exactboundaries of regulatory sequences have not been completely defined, DNAfragments of different lengths may have identical promoter activity.

A “promoter sequence” is a DNA regulatory region capable of binding RNApolymerase in a cell and initiating transcription of a downstream (3′direction) coding sequence. For purposes of defining the presentinvention, the promoter sequence is bounded at its 3′ terminus by thetranscription initiation site and extends upstream (5′ direction) toinclude the minimum number of bases or elements necessary to initiatetranscription at levels detectable above background. Within the promotersequence will be found a transcription initiation site (convenientlydefined for example, by mapping with nuclease SI), as well as proteinbinding domains (consensus sequences) responsible for the binding of RNApolymerase.

A coding sequence is “under the control” of transcriptional andtranslational control sequences in a cell when RNA polymerasetranscribes the coding sequence into mRNA, which is then trans-RNAspliced (if the coding sequence contains introns) and translated intothe protein encoded by the coding sequence.

“Transcriptional and translational control sequences” are DNA regulatorysequences, such as promoters, enhancers, terminators, and the like, thatprovide for the expression of a coding sequence in a host cell. Ineukaryotic cells, polyadenylation signals are control sequences.

The term “response element” means one or more cis-acting DNA elementswhich confer responsiveness on a promoter mediated through interactionwith the DNA binding domains of the first chimeric gene. This DNAelement may be either palindromic (perfect or imperfect) in its sequenceor composed of sequence motifs or half sites separated by a variablenumber of nucleotides. The half sites can be similar or identical andarranged as either direct or inverted repeats or as a single half siteor multimers of adjacent half sites in tandem. The response element maycomprise a minimal promoter isolated from different organisms dependingupon the nature of the cell or organism into which the response elementwill be incorporated. The DNA binding domain of the first hybrid proteinbinds, in the presence or absence of a ligand, to the DNA sequence of aresponse element to initiate or suppress transcription of downstreamgene(s) under the regulation of this response element. Examples of DNAsequences for response elements of the natural ecdysone receptorinclude: RRGG/TTCANTGAC/ACYY, (SEQ ID NO:26), (see Cherbas L., et. al.,(1991), Genes Dev. 5, 120-131); AGGTCAN_((n))AGGTCA, (SEQ ID NO:27),where N_((n)) can be one or more spacer nucleotides (see D'Avino P P.,et. al., (1995), Mol. Cell. Endocrinol, 113, 1-9); and GGGTTGAATGAATTT(SEQ ID NO:28), (see Antoniewski C., et. al., (1994). Mol. Cell Biol.14, 4465-4474).

The term “operably linked” refers to the association of nucleic acidsequences on a single nucleic acid fragment so that the function of oneis affected by the other. For example, a promoter is operably linkedwith a coding sequence when it is capable of affecting the expression ofthat coding sequence (i.e., that the coding sequence is under thetranscriptional control of the promoter). Coding sequences can beoperably linked to regulatory sequences in sense or antisenseorientation.

The term “expression”, as used herein, refers to the transcription andstable accumulation of sense (mRNA) or antisense RNA derived from anucleic acid or polynucleotide. Expression may also refer to translationof mRNA into a protein or polypeptide.

The terms “cassette”, “expression cassette” and “gene expressioncassette” refer to a segment of DNA that can be inserted into a nucleicacid or polynucleotide at specific restriction sites or by homologousrecombination. The segment of DNA comprises a polynucleotide thatencodes a polypeptide of interest, and the cassette and restrictionsites are designed to ensure insertion of the cassette in the properreading frame for transcription and translation. “Transformationcassette” refers to a specific vector comprising a polynucleotide thatencodes a polypeptide of interest and having elements in addition to thepolynucleotide that facilitate transformation of a particular host cell.Cassettes, expression cassettes, gene expression cassettes andtransformation cassettes of the invention may also comprise elementsthat allow for enhanced expression of a polynucleotide encoding apolypeptide of interest in a host cell. These elements may include, butare not limited to: a promoter, a minimal promoter, an enhancer, aresponse element, a terminator sequence, a polyadenylation sequence, andthe like.

The terms “modulate” and “modulates” mean to induce, reduce or inhibitnucleic acid or gene expression, resulting in the respective induction,reduction or inhibition of protein or polypeptide production.

The plasmids or vectors according to the invention may further compriseat least one promoter suitable for driving expression of a gene in ahost cell. The term “expression vector” means a vector, plasmid orvehicle designed to enable the expression of an inserted nucleic acidsequence following transformation into the host. The cloned gene, i.e.,the inserted nucleic acid sequence, is usually placed under the controlof control elements such as a promoter, a minimal promoter, an enhancer,or the like. Initiation control regions or promoters, which are usefulto drive expression of a nucleic acid in the desired host cell arenumerous and familiar to those skilled in the art. Virtually anypromoter capable of driving these genes is suitable for the presentinvention including but not limited to: viral promoters, bacterialpromoters, animal promoters, mammalian promoters, synthetic promoters,constitutive promoters, tissue specific promoter, developmental specificpromoters, inducible promoters, light regulated promoters; CYC1, HIS3,GAL1, GAL4, GAL10, ADH1, PGK, PHO5, GAPDH, ADC1, TRP1, URA3, LEU2, ENO,TPI, alkaline phosphatase promoters (useful for expression inSaccharomyces): AOX1 promoter (useful for expression in Pichia);β-lactamase, lac, ara, tet, trp, IP₁, IP₈, T7, tac, and trc promoters(useful for expression in Escherichia coli); light regulated-, seedspecific-, pollen specific-, ovary specific-, pathogenesis or diseaserelated-, cauliflower mosaic virus 35S, CMV 35S minimal, cassava veinmosaic virus (CsVMV), chlorophyll a/b binding protein, ribulose 1,5-bisphosphate carboxylase, shoot-specific, root specific, chitinase,stress inducible, rice tungro bacilliform virus, plant super-promoter,potato leucine aminopeptidase, nitrate reductase, mannopine synthase,nopaline synthase, ubiquitin, zein protein, and anthocyanin promoters(useful for expression in plant cells); animal and mammalian promotersknown in the art include, but are not limited to, the SV40 early (SV40e)promoter region, the promoter contained in the 3′ long terminal repeat(LTR) of Rous sarcoma virus (RSV), the promoters of the E A or majorlate promoter (MLP) genes of adenoviruses (Ad), the cytomegalovirus(CMV) early promoter, the herpes simplex virus (HSV) thymidine kinase(TK) promoter, a baculovirus IE1 promoter, an elongation factor 1 alpha(EF1) promoter, a phosphoglycerate kinase (PGK) promoter, a ubiquitin(Ubc) promoter, an albumin promoter, the regulatory sequences of themouse metallothionein-L promoter and transcriptional control regions,the ubiquitous promoters (HPRT, vimentin, α-actin, tubulin and thelike), the promoters of the intermediate filaments (desmin,neurofilaments, keratin, GFAP, and the like), the promoters oftherapeutic genes (of the MDR, CFTR or factor VIII type, and the like),pathogenesis or disease related-promoters, and promoters that exhibittissue specificity and have been utilized in transgenic animals, such asthe elastase I gene control region which is active in pancreatic acinarcells; insulin gene control region active in pancreatic beta cells,immunoglobulin gene control region active in lymphoid cells, mousemammary tumor virus control region active in testicular, breast,lymphoid and mast cells; albumin gene, Apo AI and Apo AII controlregions active in liver, alpha-fetoprotein gene control region active inliver, alpha 1-antitrypsin gene control region active in the liver,β-globin gene control region active in myeloid cells, myelin basicprotein gene control region active in oligodendrocyte cells in thebrain, myosin light chain-2 gene control region active in skeletalmuscle, and gonadotropic releasing hormone gene control region active inthe hypothalamus, pyruvate kinase promoter, villin promoter, promoter ofthe fatty acid binding intestinal protein, promoter of the smooth musclecell α-actin, and the like. In addition, these expression sequences maybe modified by addition of enhancer or regulatory sequences and thelike.

Enhancers that may be used in embodiments of the invention include butare not limited to: an SV40 enhancer, a cytomegalovirus (CMV) enhancer,an elongation factor 1 (EF1) enhancer, yeast enhancers, viral geneenhancers, and the like.

Termination control regions, i.e., terminator or polyadenylationsequences, may also be derived from various genes native to thepreferred hosts. Optionally, a termination site may be unnecessary,however, it is most preferred if included. In a preferred embodiment ofthe invention, the termination control region may be comprise or bederived from a synthetic sequence, synthetic polyadenylation signal, anSV40 late polyadenylation signal, an SV40 polyadenylation signal, abovine growth hormone (BGH) polyadenylation signal, viral terminatorsequences, or the like.

The terms “3′ non-coding sequences” or “3′ untranslated region (UTR)”refer to DNA sequences located downstream (3′) of a coding sequence andmay comprise polyadenylation [poly(A)] recognition sequences and othersequences encoding regulatory signals capable of affecting mRNAprocessing or gene expression. The polyadenylation signal is usuallycharacterized by affecting the addition of polyadenylic acid tracts tothe 3′ end of the mRNA precursor.

“Regulatory region” means a nucleic acid sequence that regulates theexpression of a second nucleic acid sequence. A regulatory region mayinclude sequences which are naturally responsible for expressing aparticular nucleic acid (a homologous region) or may include sequencesof a different origin that are responsible for expressing differentproteins or even synthetic proteins (a heterologous region). Inparticular, the sequences can be sequences of prokaryotic, eukaryotic,or viral genes or derived sequences that stimulate or represstranscription of a gene in a specific or non-specific manner and in aninducible or non-inducible manner. Regulatory regions include origins ofreplication, RNA splice sites, promoters, enhancers, transcriptionaltermination sequences, and signal sequences which direct the polypeptideinto the secretory pathways of the target cell.

A regulatory region from a “heterologous source” is a regulatory regionthat is not naturally associated with the expressed nucleic acid.Included among the heterologous regulatory regions are regulatoryregions from a different species, regulatory regions from a differentgene, hybrid regulatory sequences, and regulatory sequences which do notoccur in nature, but are designed by one having ordinary skill in theart.

“RNA transcript” refers to the product resulting from RNApolymerase-catalyzed transcription of a DNA sequence. When the RNAtranscript is a perfect complementary copy of the DNA sequence, it isreferred to as the primary transcript or it may be a RNA sequencederived from post-transcriptional processing of the primary transcriptand is referred to as the mature RNA. “Messenger RNA (mRNA)” refers tothe RNA that is without introns and that can be translated into proteinby the cell. “cDNA” refers to a double-stranded DNA that iscomplementary to and derived from mRNA. “Sense” RNA refers to RNAtranscript that includes the mRNA and so can be translated into proteinby the cell. “Antisense RNA” refers to a RNA transcript that iscomplementary to all or part of a target primary transcript or mRNA andthat blocks the expression of a target gene. The complementarity of anantisense RNA may be with any part of the specific gene transcript,i.e., at the 5′ non-coding sequence, 3′ non-coding sequence, or thecoding sequence. “Functional RNA” refers to antisense RNA, ribozyme RNA,or other RNA that is not translated yet has an effect on cellularprocesses.

A “polypeptide” is a polymeric compound comprised of covalently linkedamino acid residues. Amino acids have the following general structure:

Amino acids are classified into seven groups on the basis of the sidechain R: (1) aliphatic side chains, (2) side chains containing ahydroxylic (OH) group, (3) side chains containing sulfur atoms, (4) sidechains containing an acidic or amide group, (5) side chains containing abasic group, (6) side chains containing an aromatic ring, and (7)proline, an imino acid in which the side chain is fused to the aminogroup. A polypeptide of the invention preferably comprises at leastabout 14 amino acids.

A “protein” is a polypeptide that performs a structural or functionalrole in a living cell.

An “isolated polypeptide” or “isolated protein” is a polypeptide orprotein that is substantially free of those compounds that are normallyassociated therewith in its natural state (e.g., other proteins orpolypeptides, nucleic acids, carbohydrates, lipids). “Isolated” is notmeant to exclude artificial or synthetic mixtures with other compounds,or the presence of impurities which do not interfere with biologicalactivity, and which may be present, for example, due to incompletepurification, addition of stabilizers, or compounding into apharmaceutically acceptable preparation.

A “variant” of a polypeptide or protein is any analogue, fragment,derivative, or mutant which is derived from a polypeptide or protein andwhich retains at least one biological property of the polypeptide orprotein. Different variants of the polypeptide or protein may exist innature. These variants may be allelic variations characterized bydifferences in the nucleotide sequences of the structural gene codingfor the protein, or may involve differential splicing orpost-translational modification. The skilled artisan can producevariants having single or multiple amino acid substitutions, deletions,additions, or replacements. These variants may include, inter alia: (a)variants in which one or more amino acid residues are substituted withconservative or non-conservative amino acids, (b) variants in which oneor more amino acids are added to the polypeptide or protein, (c)variants in which one or more of the amino acids includes a substituentgroup, and (d) variants in which the polypeptide or protein is fusedwith another polypeptide such as serum albumin. The techniques forobtaining these variants, including genetic (suppressions, deletions,mutations, etc.), chemical, and enzymatic techniques, are known topersons having ordinary skill in the art.

A “heterologous protein” refers to a protein not naturally produced inthe cell.

A “mature protein” refers to a post-translationally processedpolypeptide; i.e., one from which any pre- or propeptides present in theprimary translation product have been removed. “Precursor” proteinrefers to the primary product of translation of mRNA; i.e., with pre-and propeptides still present. Pre- and propeptides may be but are notlimited to intracellular localization signals.

The term “signal peptide” refers to an amino terminal polypeptidepreceding the secreted mature protein. The signal peptide is cleavedfrom and is therefore not present in the mature protein. Signal peptideshave the function of directing and translocating secreted proteinsacross cell membranes. Signal peptide is also referred to as signalprotein.

A “signal sequence” is included at the beginning of the coding sequenceof a protein to be expressed on the surface of a cell. This sequenceencodes a signal peptide, N-terminal to the mature polypeptide thatdirects the host cell to translocate the polypeptide. The term“translocation signal sequence” is used herein to refer to this sort ofsignal sequence. Translocation signal sequences can be found associatedwith a variety of proteins native to eukaryotes and prokaryotes, and areoften functional in both types of organisms.

The term “homology” refers to the percent of identity between twopolynucleotide or two polypeptide moieties. The correspondence betweenthe sequence from one moiety to another can be determined by techniquesknown to the art. For example, homology can be determined by a directcomparison of the sequence information between two polypeptide moleculesby aligning the sequence information and using readily availablecomputer programs. Alternatively, homology can be determined byhybridization of polynucleotides under conditions that form stableduplexes between homologous regions, followed by digestion withsingle-stranded-specific nuclease(s) and size determination of thedigested fragments.

As used herein, the term “homologous” in all its grammatical forms andspelling variations refers to the relationship between proteins thatpossess a “common evolutionary origin,” including proteins fromsuperfamilies (e.g., the immunoglobulin superfamily) and homologousproteins from different species (e.g., myosin light chain, etc.) (Reecket al., 1987, Cell 50: 667). Such proteins (and their encoding genes)have sequence homology, as reflected by their high degree of sequencesimilarity. However, in common usage and in the instant application, theterm “homologous,” when modified with an adverb such as “highly,” mayrefer to sequence similarity and not a common evolutionary origin.

Accordingly, the term “sequence similarity” in all its grammatical formsrefers to the degree of identity or correspondence between nucleic acidor amino acid sequences of proteins that may or may not share a commonevolutionary origin (see Reeck et al., 1987, Cell 50:667).

In a specific embodiment, two DNA sequences are “substantiallyhomologous” or “substantially similar” when at least about 50%(preferably at least about 75%, and most preferably at least about 90 or95%) of the nucleotides match over the defined length of the DNAsequences. Sequences that are substantially homologous can be identifiedby comparing the sequences using standard software available in sequencedata banks, or in a Southern hybridization experiment under, forexample, stringent conditions as defined for that particular system.Defining appropriate hybridization conditions is within the skill of theart. See, e.g., Sambrook et al., 1989, supra.

As used herein, “substantially similar” refers to nucleic acid fragmentswherein changes in one or more nucleotide bases results in substitutionof one or more amino acids, but do not affect the functional propertiesof the protein encoded by the DNA sequence. “Substantially similar” alsorefers to nucleic acid fragments wherein changes in one or morenucleotide bases does not affect the ability of the nucleic acidfragment to mediate alteration of gene expression by antisense orco-suppression technology. “Substantially similar” also refers tomodifications of the nucleic acid fragments of the instant inventionsuch as deletion or insertion of one or more nucleotide bases that donot substantially affect the functional properties of the resultingtranscript. It is therefore understood that the invention encompassesmore than the specific exemplary sequences. Each of the proposedmodifications is well within the routine skill in the art, as isdetermination of retention of biological activity of the encodedproducts.

Moreover, the skilled artisan recognizes that substantially similarsequences encompassed by this invention are also defined by theirability to hybridize, under stringent conditions (0.1×SSC, 0.1% SDS, 65°C. and washed with 2×SSC, 0.1% SDS followed by 0.1×SSC, 0.1% SDS), withthe sequences exemplified herein. Substantially similar nucleic acidfragments of the instant invention are those nucleic acid fragmentswhose DNA sequences are at least 70% identical to the DNA sequence ofthe nucleic acid fragments reported herein. Preferred substantiallynucleic acid fragments of the instant invention are those nucleic acidfragments whose DNA sequences are at least 80% identical to the DNAsequence of the nucleic acid fragments reported herein. More preferrednucleic acid fragments are at least 90% identical to the DNA sequence ofthe nucleic acid fragments reported herein. Even more preferred arenucleic acid fragments that are at least 95% identical to the DNAsequence of the nucleic acid fragments reported herein.

Two amino acid sequences are “substantially homologous” or“substantially similar” when greater than about 40% of the amino acidsare identical, or greater than 60% are similar (functionally identical).Preferably, the similar or homologous sequences are identified byalignment using, for example, the GCG (Genetics Computer Group, ProgramManual for the GCG Package, Version 7, Madison, Wis.) pileup program.

The term “corresponding to” is used herein to refer to similar orhomologous sequences, whether the exact position is identical ordifferent from the molecule to which the similarity or homology ismeasured. A nucleic acid or amino acid sequence alignment may includespaces. Thus, the term “corresponding to” refers to the sequencesimilarity, and not the numbering of the amino acid residues ornucleotide bases.

A “substantial portion” of an amino acid or nucleotide sequencecomprises enough of the amino acid sequence of a polypeptide or thenucleotide sequence of a gene to putatively identify that polypeptide orgene, either by manual evaluation of the sequence by one skilled in theart, or by computer-automated sequence comparison and identificationusing algorithms such as BLAST (Basic Local Alignment Search Tool;Altschul. S. F., et al., (1993) J. Mol. Biol. 215: 403-410; see alsowww.ncbi.nlm.nih.gov/BLAST/). In general, a sequence often or morecontiguous amino acids or thirty or more nucleotides is necessary inorder to putatively identify a polypeptide or nucleic acid sequence ashomologous to a known protein or gene. Moreover, with respect tonucleotide sequences, gene specific oligonucleotide probes comprising20-30 contiguous nucleotides may be used in sequence-dependent methodsof gene identification (e.g., Southern hybridization) and isolation(e.g., in situ hybridization of bacterial colonies or bacteriophageplaques). In addition, short oligonucleotides of 12-15 bases may be usedas amplification primers in PCR in order to obtain a particular nucleicacid fragment comprising the primers. Accordingly, a “substantialportion” of a nucleotide sequence comprises enough of the sequence tospecifically identify and/or isolate a nucleic acid fragment comprisingthe sequence.

The term “percent identity”, as known in the art, is a relationshipbetween two or more polypeptide sequences or two or more polynucleotidesequences, as determined by comparing the sequences. In the art,“identity” also means the degree of sequence relatedness betweenpolypeptide or polynucleotide sequences, as the case may be, asdetermined by the match between strings of such sequences. “Identity”and “similarity” can be readily calculated by known methods, includingbut not limited to those described in: Computational Molecular Biology(Lesk, A. M., ed.) Oxford University Press, New York (1988);Biocomputing: Informatics and Genome Projects (Smith, D. W., ed.)Academic Press, New York (1993); Computer Analysis of Sequence Data,Part 1 (Griffin, A. M., and Griffin, H. G., eds.) Humana Press, NewJersey (1994): Sequence Analysis in Molecular Biology (von Heinje, G.,ed.) Academic Press (1987); and Sequence Analysis Primer (Gribskov, M.and Devereux, J., eds.) Stockton Press, New York (1991). Preferredmethods to determine identity are designed to give the best matchbetween the sequences tested. Methods to determine identity andsimilarity are codified in publicly available computer programs.Sequence alignments and percent identity calculations may be performedusing the Megalign program of the LASERGENE bioinformatics computingsuite (DNASTAR Inc., Madison, Wis.). Multiple alignments of thesequences may be performed using the Clustal method of alignment(Higgins and Sharp (1989) CABIOS. 5:151-153) with the default parameters(GAP PENALTY=1O, GAP LENGTH PENALTY=10). Default parameters for pairwisealignments using the Clustal method may be selected: KTUPLE 1, GAPPENALTY=3, WINDOW=5 and DIAGONALS SAVED=5.

The term “sequence analysis software” refers to any computer algorithmor software program that is useful for the analysis of nucleotide oramino acid sequences. “Sequence analysis software” may be commerciallyavailable or independently developed. Typical sequence analysis softwarewill include but is not limited to the GCG suite of programs (WisconsinPackage Version 9.0, Genetics Computer Group (GCG), Madison, Wis.),BLASTP, BLASTN, BLASTX (Altschul et al., J. Mol. Biol. 215: 403-410(1990), and DNASTAR (DNASTAR, Inc. 1228 S. Park St. Madison, Wis. 53715USA). Within the context of this application it will be understood thatwhere sequence analysis software is used for analysis, that the resultsof the analysis will be based on the “default values” of the programreferenced, unless otherwise specified. As used herein “default values”will mean any set of values or parameters, which originally load withthe software when first initialized.

“Synthetic genes” can be assembled from oligonucleotide building blocksthat are chemically synthesized using procedures known to those skilledin the art. These building blocks are ligated and annealed to form genesegments that are then enzymatically assembled to construct the entiregene. “Chemically synthesized”, as related to a sequence of DNA, meansthat the component nucleotides were assembled in vitro. Manual chemicalsynthesis of DNA may be accomplished using well-established procedures,or automated chemical synthesis can be performed using one of a numberof commercially available machines. Accordingly, the genes can betailored for optimal gene expression based on optimization of nucleotidesequence to reflect the codon bias of the host cell. The skilled artisanappreciates the likelihood of successful gene expression if codon usageis biased towards those codons favored by the host. Determination ofpreferred codons can be based on a survey of genes derived from the hostcell where sequence information is available.

The Invention

The present invention provides compositions and methods for obtainingsite-specific recombination in eukaryotic cells. More specifically, theinvention employs prokaryotic recombinases, such as bacteriophagerecombinases, that are unidirectional in that they can catalyzerecombination between two complementary recombination sites, but cannotcatalyze recombination between the hybrid sites that are formed by thisrecombination. The inventor has identified novel recombinases that eachdirects recombination only between a bacterial attachment site (attB)and a phage attachment site (attP). The recombinase cannot mediaterecombination between the attL, and attR hybrid sites that are formedupon recombination between attB and attP. Because recombinases such asthese cannot alone catalyze the reverse reaction, the attB and attPrecombination is stable. This property is one that sets the compositionsand methods of the present invention apart from other recombinationsystems currently used for eukaryotic cells, such as the Cre-lox orFLP-FRT system, where the recombination reactions are reversible. Use ofthe recombination systems of the present invention provides newopportunities for directing stable transgene and chromosomerearrangements in eukaryotic cells.

The methods of the present invention involve contacting a pair ofrecombination attachment sites, attB and attP, that are present in aeukaryotic cell with a corresponding recombinase. The recombinase thenmediates recombination between the recombination attachment sites.Depending upon the relative locations of the recombination attachmentsites, any one of a number of events can occur as a result of therecombination. For example, if the recombination attachment sites arepresent on different nucleic acid molecules, the recombination canresult in integration of one nucleic acid molecule into a secondmolecule. Thus, one can obtain integration of a plasmid that containsone recombination site into a eukaryotic cell chromosome that includesthe corresponding recombination site. Because the recombinases used inthe methods of the invention cannot catalyze the reverse reaction, theintegration is stable. Such methods are useful, for example, forobtaining stable integration into the eukaryotic chromosome of atransgene that is present on the plasmid.

The recombination attachment sites can also be present on the samenucleic acid molecule. In such cases, the resulting product typicallydepends upon the relative orientation of the attachment sites. Forexample, recombination between sites that are in the parallel or directorientation will generally result in excision of any DNA that liesbetween the recombination attachment sites. In contrast, recombinationbetween attachment sites that are in the reverse orientation can resultin inversion of the intervening DNA. Likewise, the resulting rearrangednucleic acid is stable in that the recombination is irreversible in theabsence of an additional factor or factors, generally encoded by theparticular bacteriophage and/or by the host cell of the bacteriophagefrom which the recombinase is derived, that is not normally found ineukaryotic cells. One example of an application for which this method isuseful involves the placement of a promoter between the recombinationattachment sites. If the promoter is initially in the oppositeorientation relative to a coding sequence that is to be expressed by thepromoter and the recombination sites that flank the promoter are in theinverted orientation, contacting the recombination attachment sites willresult in inversion of the promoter, thus placing the promoter in thecorrect orientation to drive expression of the coding sequence.Similarly, if the promoter is initially in the correct orientation forexpression and the recombination attachment sites are in the sameorientation, contacting the recombination attachment sites with therecombinase can result in excision of the promoter fragment, thusstopping expression of the coding sequence.

The methods of the invention are also useful for obtainingtranslocations of chromosomes. For example, in these embodiments, onerecombination attachment site is placed on one chromosome and a secondrecombination attachment site that can serve as a substrate forrecombination with the first recombination attachment site is placed ona second chromosome. Upon contacting the recombination attachment siteswith a recombinase, recombination occurs that results in swapping of thetwo chromosome arms. For example, one can construct two strains of anorganism, one strain of which includes the first recombinationattachment site and the second strain that contains the secondrecombination attachment site. The two strains are then crossed, toobtain a progeny strain that includes both of the recombinationattachment sites. Upon contacting the attachment sites with therecombinase, chromosome arm swapping occurs.

Recombinases

The recombinases used in the practice of the present invention can beintroduced into a target cell before, concurrently with, or after theintroduction of a targeting vector. The recombinase can be directlyintroduced into a cell as a protein, for example, using liposomes,coated particles, or microinjection. Alternately, a polynucleotide,either DNA or messenger RNA, encoding the recombinase can be introducedinto the cell using a suitable expression vector. The targeting vectorcomponents described above are useful in the construction of expressioncassettes containing sequences encoding a recombinase of interest.However, expression of the recombinase can be regulated in other ways,for example, by placing the expression of the recombinase under thecontrol of a regulatable promoter (i.e., a promoter whose expression canbe selectively induced or repressed).

Recombinases for use in the practice of the present invention can beproduced recombinantly or purified as previously described. Polypeptideshaving the desired recombinase activity can be purified to a desireddegree of purity by methods known in the art of protein ammonium sulfateprecipitation, purification, including, but not limited to, sizefractionation, affinity chromatography, HPLC, ion exchangechromatography, heparin agarose affinity chromatography (e.g., Thorpe &Smith, Proc. Nat. Acad. Sci. 95:5505-5510, 1998.)

Recombinase polypeptides, and nucleic acids that encode the recombinasepolypeptides of the present invention, are described in Example 1, andcan be obtained using routine methods known to those of skill in theart. In preferred embodiments the recombinase is an isolatedpolynucleotide sequence comprising a nucleic acid that is at least 90%identical to the nucleic acid sequence selected from the groupconsisting of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7,and SEQ ID NO: 9, wherein the nucleic acid has recombinase activity.More preferably the recombinase is an isolated polynucleotide sequencecomprising the nucleic acid sequence selected from the group consistingof SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, and SEQ IDNO: 9. Even more preferably the recombinase is an isolatedpolynucleotide sequence comprising a nucleic acid sequence that encodesa recombinase selecting from the group consisting of a SPβc2recombinase, a SF370.1 recombinase, a Bxb1 recombinase, an A118recombinase and a φRv1 recombinase.

The recombinases can be introduced into the eukaryotic cells thatcontain the recombination attachment sites at which recombination isdesired by any suitable method. Methods of introducing functionalproteins, e.g., by microinjection or other methods, into cells are wellknown in the art. Introduction of purified recombinase protein ensures atransient presence of the protein and its function, which is often apreferred embodiment. Alternatively, a gene encoding the recombinase canbe included in an expression vector used to transform the cell, in whichthe recombinase-encoding polynucleotide is operably linked to a promoterwhich mediates expression of the polynucleotide in the eukaryotic cell.The recombinase polypeptide can also be introduced into the eukaryoticcell by messenger RNA that encodes the recombinase polypeptide. It isgenerally preferred that the recombinase be present for only such timeas is necessary for insertion of the nucleic acid fragments into thegenome being modified. Thus, the lack of permanence associated with mostexpression vectors is not expected to be detrimental. One can introducethe recombinase gene into the cell before, after, or simultaneouslywith, the introduction of the exogenous polynucleotide of interest. Inone embodiment, the recombinase gene is present within the vector thatcarries the polynucleotide that is to be inserted; the recombinase genecan even be included within the polynucleotide. In other embodiments,the recombinase gene is introduced into a transgenic eukaryoticorganism, e.g., a transgenic plant, animal, fungus, or the like, whichis then crossed with an organism that contains the correspondingrecombination sites. Transgenic cells or animals can be made thatexpress a recombinase constitutively or under cell-specific,tissue-specific, developmental-specific, organelle-specific, or smallmolecule-inducible or repressible promoters. The recombinases can bealso expressed as a fusion protein with other peptides, proteins,nuclear localizing signal peptides, signal peptides, ororganelle-specific signal peptides (e.g., mitochondrial or chloroplasttransit peptides to facilitate recombination in mitochondria orchloroplast).

In embodiments of the present invention, recombination attachment sitescomprise an isolated polynucleotide sequence comprising a nucleic acidthat is at least 90% identical to the nucleic acid sequence selectedfrom the group consisting of SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO:14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ IDNO: 19, SEQ ID NO: 20, and SEQ ID NO: 21. Preferably the attachment siteis an isolated polynucleotide sequence comprising the nucleic acidsequence selected from the group consisting of SEQ ID NO: 11, SEQ ID NO:13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ IDNO: 18, SEQ ID NO: 19, SEQ ID NO: 20, and SEQ ID NO: 21.

Vectors/Constructs

The targeting constructs contemplated by the invention may containadditional nucleic acid fragments such as control sequences, markersequences, selection sequences and the like as discussed below.

The present invention also provides means for targeted insertion of apolynucleotide (or nucleic acid sequence(s)) of interest into a genomeby, for example, (i) providing a recombinase, wherein the recombinase iscapable of facilitating recombination between a first recombination siteand a second recombination site, (ii) providing a targeting constructhaving a first recombination sequence and a polynucleotide of interest,(iii) introducing the recombinase and the targeting construct into acell which contains in its nucleic acid the second recombination site,wherein said introducing is done under conditions that allow therecombinase to facilitate a recombination event between the first andsecond recombination sites.

The present invention also relates to a vector for site-specificintegration of a polynucleotide sequence into the genome of an isolatedeukaryotic cell, said vector comprising a polynucleotide of interest,and a second recombination attB or attP site, wherein said secondrecombination attB or attP site comprises a polynucleotide sequence thatrecombines with a first recombination attP or attB site or pseudo attPor pseudo attB site in the genome of said isolated eukaryotic cell andsaid recombination occurs in the presence of a site-specific recombinaseselected from the group consisting of a Listeria monocytogenes phagerecombinase, a Streptococcus pyogenes phage recombinase, a Bacillussubtilis phage recombinase, a Mycobacterium tuberculosis phagerecombinase and a Mycobacterium smegmatis phage recombinase, providedthat when the first recombination site is attB or pseudo attB, thesecond recombination site is attP and when the first recombination siteis attP or pseudo attP, the second recombination site is attB.Preferably the recombinase is selected from the group consisting of anA118 recombinase, a SF370.1 recombinase, a SPβc2 recombinase, a φRv1recombinase, and a Bxb1 recombinase.

Polynucleotides of interest can include, but are not limited to,expression cassettes encoding polypeptide products. The targetingconstructs can be circular or linear and may also contain selectablemarkers, an origin of replication, and other elements.

A variety of expression vectors are suitable for use in the practice ofthe present invention, both for prokaryotic expression and eukaryoticexpression. In general, the targeting construct will have one or more ofthe following features: a promoter, promoter-enhancer sequences, aselection marker sequence, an origin of replication, an inducibleelement sequence, an epitope-tag sequence, and the like.

Promoter and promoter-enhancer sequences are DNA sequences to which RNApolymerase binds and initiates transcription. The promoter determinesthe polarity of the transcript by specifying which strand will betranscribed. Bacterial promoters consist of consensus sequences, −35 and−10 nucleotides relative to the transcriptional start, which are boundby a specific sigma factor and RNA polymerase. Eukaryotic promoters aremore complex. Most promoters utilized in expression vectors aretranscribed by RNA polymerase II. General transcription factors (GTFS)first bind specific sequences near the start and then recruit thebinding of RNA polymerase II. In addition to these minimal promoterelements, small sequence elements are recognized specifically by modularDNA-binding/trans-activating proteins (e.g. AP-1, SP-1) that regulatethe activity of a given promoter. Viral promoters serve the samefunction as bacterial or eukaryotic promoters and either provide aspecific RNA polymerase in trans (bacteriophage T7) or recruit cellularfactors and RNA polymerase (SV40, RSV, CMV). Viral promoters may bepreferred as they are generally particularly strong promoters.

Promoters may be, furthermore, either constitutive or regulatable (i.e.,inducible or repressible). Inducible elements are DNA sequence elementswhich act in conjunction with promoters and bind either repressors (e.g.lacO/LAC 1q repressor system in E. coli) or inducers (e.g. gall/GALAinducer system in yeast). In either case, transcription is virtually“shut off” until the promoter is repressed or induced, at which pointtranscription is “turned-on.”

Examples of constitutive promoters include the int promoter ofbacteriophage λ, the bla promoter of the β-lactarmase gene sequence ofpBR322, the CAT promoter of the chloramphenicol acetyl transferase genesequence of pPR325, and the like. Examples of inducible prokaryoticpromoters include the major right and left promoters of bacteriophage(P.sub.L and P.sub.R), the trp, reca, lacZ, AraC and gal promoters of E.coli, the α-amylase (Ulmanen Ett at., J. Bacteriol. 162:176-182, 1985)and the sigma-28-specific promoters of B. subtilis (Gilman et al., Genesequence 32:11-20(1984)), the promoters of the bacteriophages ofBacillus (Gryczan, In: The Molecular Biology of the Bacilli, AcademicPress, Inc., NY (1982)), Streptomyes promoters (Ward et al., Mol. Gen.Genet. 203:468-478, 1986), and the like. Exemplary prokaryotic promotersare reviewed by Glick (J. Ind. Microtiot. 1:277-282, 1987); Cenatiempo(Biochimie 68: 505-516, 1986); and Gottesman (Ann. Rev. Genet.18:415-442, 1984).

Preferred eukaryotic promoters include, but are not limited to, thefollowing: the promoter of the mouse metallothionein I gene sequence(Hamer et al., J. Mol. Appl. Gen. 1:273-288, 1982); the TK promoter ofHerpes virus (McKnight, Cell 31:355-365, 1982); the SV40 early promoter(Benoist et al., Nature (London) 290:304-310, 1981); the yeast gall genesequence promoter (Johnston et al., Proc. Natl. Acad. Sci. (USA)79:6971-6975, 1982); Silver et al., Proc. Natl. Acad. Sci. (USA)81:5951-59SS, 1984), the CMV promoter, the EF-1 promoter,ecdysone-responsive promoter(s), tetracycline-responsive promoter, andthe like. Exemplary promoters for use in the present invention areselected such that they are functional in cell type (and/or animal orplant) into which they are being introduced.

Selection markers are valuable elements in expression vectors as theyprovide a means to select for growth of only those cells that contain avector. Such markers are of two types: drug resistance and auxotrophic.A drug resistance marker enables cells to detoxify an exogenously addeddrug that would otherwise kill the cell. Auxotrophic markers allow cellsto synthesize an essential component (usually an amino acid) while grownin media that lacks that essential component.

Common selectable marker genes include those for resistance toantibiotics such as ampicillin, tetracycline, kanamycin, bleomycin,streptomycin, hygromycin, neomycin, Zeocin™, and the like. Selectableauxotrophic genes include, for example, hisD, that allows growth inhistidine free media in the presence of histidinol.

A further element useful in an expression vector is an origin ofreplication. Replication origins are unique DNA segments that containmultiple short repeated sequences that are recognized by multimericorigin-binding proteins and that play a key role in assembling DNAreplication enzymes at the origin site. Suitable origins of replicationfor use in expression vectors employed herein include E. coli oriC,colE1 plasmid origin, 2μ and ARS (both useful in yeast systems), sfl,SV40, EBV oriP (useful in mammalian systems), and the like.

Epitope tags are short peptide sequences that are recognized by epitopespecific antibodies. A fusion protein comprising a recombinant proteinand an epitope tag can be simply and easily purified using an antibodybound to a chromatography resin. The presence of the epitope tagfurthermore allows the recombinant protein to be detected in subsequentassays, such as Western blots, without having to produce an antibodyspecific for the recombinant protein itself. Examples of commonly usedepitope tags include V5, glutathione-S-transferase (GST), hemaglutinin(HA), the peptide Phe-His-His-Thr-Thr, chitin binding domain, and thelike.

A further useful element in an expression vector is a multiple cloningsite or polylinker. Synthetic DNA encoding a series of restrictionendonuclease recognition sites is inserted into a plasmid vector, forexample, downstream of the promoter element. These sites are engineeredfor convenient cloning of DNA into the vector at a specific position.

The foregoing elements can be combined to produce expression vectorssuitable for use in the methods of the invention. Those of skill in theart would be able to select and combine the elements suitable for use intheir particular system in view of the teachings of the presentspecification. Suitable prokaryotic vectors include plasmids such asthose capable of replication in E. coli (for example, pBR322, ColE1,pSC101, PACYC 184, itVX, PRSET, pBAD (Invitrogen, Carlsbad, Calif.) andthe like). Such plasmids are disclosed by Sambrook (cf. “MolecularCloning: A Laboratory Manual,” second edition, edited by Sambrook,Fritsch, & Maniatis, Cold Spring Harbor Laboratory, (1989)). Bacillusplasmids include pC194, pC221, pT127, and the like, and are disclosed byGryczan (In: The Molecular Biology of the Bacilli, Academic Press, NY(1982), pp. 307-329). Suitable Streptomyces plasmids include pli101(Kendall et al., J. Bacteriol. 169:4177-4183, 1987), and Streptomycesbacteriophages such as φC31 (Chater et al., In: Sixth InternationalSymposium on Actinomycetales Biology, Akademiai Kaido, Budapest, Hungary(1986), pp. 45-54). Pseudomonas plasmids are reviewed by John et al.(Rev. Infect. Dis. 8:693-704, 1986), and Izaki (Jpn. J. Bacteriol.33:729-742, 1978).

Suitable eukaryotic plasmids include, for example, BPV, EBV, vaccinia,SV40, 2-micron circle, pcDNA3.1, pcDNA3.1/GS, pDual, pYES2/GS, pMT, pIND, pIND (Spl), pVgRXR (Invitrogen), and the like, or theirderivatives. Such plasmids are well known in the art (Botstein et al.,Miami Wntr. SyTnp. 19:265-274, 1982; Broach, In: “The Molecular Biologyof the Yeast Saccharomyces: Life Cycle and Inheritance”, Cold SpringHarbor Laboratory, Cold Spring Harbor. N.Y., p. 445-470, 1981; Broach,Cell 28:203-204, 1982; Dilon et al., J. Clin. Hematol. Oncol. 10: 39-48,1980; Maniatis, In: Cell Biology: A Comprehensive Treatise, Vol. 3, GeneSequence Expression, Academic Press, NY, pp. 563-608, 1980. Thetargeting cassettes described herein can be constructed utilizingmethodologies known in the art of molecular biology (see, for example,Ausubel or Maniatis) in view of the teachings of the specification. Asdescribed above, the targeting constructs are assembled by inserting,into a suitable vector backbone, a recombination attachment site,polynucleotides encoding sequences of interest operably linked to apromoter of interest; and, optionally a sequence encoding a positiveselection marker.

A preferred method of obtaining polynucleotides, including suitableregulatory sequences (e.g., promoters) is PCR. General procedures forPCR are taught in MacPherson et al., PCR: A PRACTICAL APPROACH, (IRLPress at Oxford University Press, (1991)). PCR conditions for eachapplication reaction may be empirically determined. A number ofparameters influence the success of a reaction. Among these parametersare annealing temperature and time, extension time, Mg²⁺ and ATPconcentration, pH, and the relative concentration of primers, templatesand deoxyribonucleotides. After amplification, the resulting fragmentscan be detected by agarose gel electrophoresis followed by visualizationwith ethidium bromide staining and ultraviolet illumination.

The expression cassettes, targeting constructs, vectors, recombinasesand recombinase-coding sequences of the present invention can beformulated into kits. Components of such kits can include, but are notlimited to, containers, instructions, solutions, buffers, disposablesand hardware.

Methods

The present invention relates to a method for site-specificrecombination comprising: providing a first recombination site and asecond recombination site; contacting the first and second recombinationsites with a prokaryotic recombinase polypeptide, resulting inrecombination between the recombination sites, wherein the recombinasepolypeptide can mediate recombination between the first and secondrecombination sites, the first recombination site is attP or attB, thesecond recombination site is attB or attP, and the recombinase isselected from the group consisting of a Listeria monocytogenes phagerecombinase, a Streptococcus pyogenes phage recombinase, a Bacillussubtilis phage recombinase, a Mycobacterium tuberculosis phagerecombinase and a Mycobacterium smegmatis phage recombinase, providedthat when the first recombination attachment site is attB, the secondrecombination attachment site is attP. and when the first recombinationattachment site is attP. the second recombination attachment site isattB.

Further methods of the present invention provide for the introduction ofa site-specific recombinase into a cell whose genome is to be modified.A preferred embodiment of the present invention relates to a method forobtaining site-specific recombination in a eukaryotic cell comprisesproviding a eukaryotic cell that comprises a first recombinationattachment site and a second recombination attachment site; contactingthe first and second recombination attachment sites with a prokaryoticrecombinase polypeptide, resulting in recombination between therecombination attachment sites, wherein the recombinase polypeptide canmediate recombination between the first and second recombinationattachment sites, the first recombination attachment site is a phagegenomic recombination attachment site (attP) or a bacterial genomicrecombination attachment site (attB), the second recombinationattachment site is attB or attP, and the recombinase is selected fromthe group consisting of a Listeria monocytogenes phage recombinase, aStreptococcus pyogenes phage recombinase, a Bacillus subtilis phagerecombinase, a Mycobacterium tuberculosis phage recombinase and aMycobacterium smegmatis phage recombinase, provided that when the firstrecombination attachment site is attB, the second recombinationattachment site is attP, and when the first recombination attachmentsite is attP, the second recombination attachment site is attB. In apreferred embodiment the recombinase is selected from the groupconsisting of an A118 recombinase, a SF370.1 recombinase, a SPβc2recombinase, a φRv1 recombinase, and a Bxb1 recombinase. In oneembodiment the recombination results in integration. Targetedintegration of transgenes into predefined genetic loci is a desirablegoal for many applications. First, a first recombination site for asite-specific recombinase is inserted at a genomic site, either at arandom or at a predetermined location. Subsequently, the cells aretransfected with a plasmid carrying the gene or DNA of interest and thesecond recombination site and a source for recombinase (expressionplasmid, RNA, protein, or virus-expressing recombinase). Recombinationbetween the first and second recombination sites leads to integration ofplasmid DNA.

In another embodiment the site-specific recombination results in adeletion or excision. The most common application in mammalian geneticsis the inactivation or activation at a defined developmental stage. TheDNA or gene to be deleted or excised from the chromosomes or episomalDNA is flanked by tandem (direct) repeats of first recombination andsecond recombination sites. Recombination between the sites due to theintroduction of a recombinase leads to deletion of the DNA and geneinactivation. In another type of application, a recombinase can mediateexcision of a transcriptional stop signal (present between the promoterand gene) from the genome, thereby linking the promoter element to theopen reading frame of a transgene and activating gene expression. Therecombinase can be expressed using a constitutive or inducible promoteror by introducing a recombinase-expressing viral vector.

In an additional embodiment, the site-specific recombination results inan inversion. Recombination between first and second recombination sitesinserted into the same DNA molecule (intramolecular recombination) inopposite orientations leads to inversion of the intervening DNA segmentor fragment.

In a further embodiment, the site-specific recombination results in anexchange of DNA. First a cassette acceptor is created at a location ofinterest in the chromosome. The cassette acceptor contains DNA ofinterest, very often a selectable marker gene flanked on either side byfirst recombination site (for example, attB). Second, an exchange vectorcontaining replacement DNA cassette flanked on either side by therecombination site (for example, attP) is introduced into cells alongwith the recombinase expression plasmid or recombinase protein. Doublecross between the cognate recombination recognition sites leads to thereplacement of the DNA between the first recombination sites with thatcarried by the exchange vector. In another instance, the firstrecombination site is attP and second recombination site is attB. Thisprocedure is often called recombinase-mediated cassette exchange.

In an additional embodiment, the site-specific recombination results inchromosomal translocations. For chromosomal translocation, a firstrecombination site is introduced into a first chromosome and secondrecombination site is introduced into a second chromosome. Supplying thecells with a recombinase leads to translocation of the chromosomes.Translocations are generated when recombination sites are targeted tonon-homologous chromosomes. Depending on the relative orientation ofrecombinase sites, recombination leads to translocation or dicentric andacentric chromosomes. When the recombination sites are oriented in thedirection relative to their respective centromeres, translocationoccurs. If the recombination sites are in opposite orientation,recombination will result in acentric and dicentric chromosomes.

The present invention also comprises recombinase-mediated DNA insertionat pseudo recombination attachment sites present in the genome. Pseudorecombination or attachment site of the specific recombinase is a nativesequence present on the chromosome that the site-specific recombinasecan recognize and use for integrating of plasmid DNA containing thefirst or second recombination sites. The integration at pseudorecombination site is often more frequent than the random integration.This is a one step process in the sense that there is no need tointroduce a recombination site into the genome as a first step.Integration at pseudo-sites has applications in gene and cell therapy.Pseudo attB is a native recombination site present in the genome thatrecombines with attP site. Pseudo attP is a native recombination sitepresent in the genome that recombines with attB site. Accordingly, thepresent invention provides for a method for obtaining site-specificrecombination in a eukaryotic cell, the method comprising: providing aeukaryotic cell that comprises a first recombination site and a secondrecombination site; contacting the first and second recombination siteswith a prokaryotic recombinase polypeptide, resulting in recombinationbetween the recombination sites, wherein the recombinase polypeptide canmediate recombination between the first and second recombination sites,the first recombination site is attP or attB, the second recombinationsite is a pseudo attachment site, and the recombinase is selected fromthe group consisting of a Listeria monocytogenes phage recombinase, aStreptococcus pyogenes phage recombinase, a Bacillus subtilis phagerecombinase, a Mycobacterium tuberculosis phage recombinase and aMycobacterium smegmatis phage recombinase. Preferably the recombinase isselected from the group consisting of an A118 recombinase, a SF370.1recombinase, a SPβc2 recombinase, a φRv1 recombinase, and a Bxb1recombinase.

The present invention further comprises methods for obtaining aeukaryotic cell having a stably integrated polynucleotide sequence, themethod comprising: introducing a polynucleotide into a eukaryotic cellthat comprises a first recombination attB or attP site, wherein thepolynucleotide comprises a nucleic acid sequence and a secondrecombination attP or attB site, and contacting the first and the secondrecombination sites with a prokaryotic recombinase polypeptide, whereinthe recombinase polypeptide can mediate site-specific recombinationbetween the first and second recombination sites, and the recombinase isselected from the group consisting of a Listeria monocytogenes phagerecombinase, a Streptococcus pyogenes phage recombinase, a Bacillussubtilis phage recombinase, a Mycobacterium tuberculosis phagerecombinase and a Mycobacterium smegmatis phage recombinase, providedthat when the first recombination site is attB, the second recombinationsite is attP and when the first recombination site is attP, the secondrecombination site is attB. In another embodiment the method forobtaining a eukaryotic cell having a stably integrated polynucleotidesequence comprises: introducing a polynucleotide into a eukaryotic cellthat comprises a first recombination pseudo attachment site, wherein thepolynucleotide comprises a nucleic acid sequence and a secondrecombination attP or attB site, and contacting the first and the secondrecombination sites with a prokaryotic recombinase polypeptide, whereinthe recombinase polypeptide can mediate site-specific recombinationbetween the first and second recombination sites, and the recombinase isselected from the group consisting of a Listeria monocytogenes phagerecombinase, a Streptococcus pyogenes phage recombinase, a Bacillussubtilis phage recombinase, a Mycobacterium tuberculosis phagerecombinase and a Mycobacterium smegmatis phage recombinase. Inpreferred embodiments the recombinase is selected from the groupconsisting of an A118 recombinase, a SF370.1 recombinase, a SPβc2recombinase, a φRv1 recombinase, and a Bxb1 recombinase.

The present invention additionally comprises a method for obtainingsite-specific recombination in a eukaryotic cell, the method comprising:providing a eukaryotic cell that comprises a first recombination siteand a second recombination site with a polynucleotide sequence flankedby a third recombination site and a fourth recombination site;contacting the recombination sites with a prokaryotic recombinasepolypeptide, resulting in recombination between the recombination sites,wherein the recombinase polypeptide can mediate recombination betweenthe first and third recombination sites and the second and fourthrecombination sites, the first and second recombination sites are attPor attB, the third and fourth recombination sites are attB or attP, andthe recombinase is selected from the group consisting of a Listeriamonocytogenes phage recombinase, a Streptococcus pyogenes phagerecombinase, a Bacillus subtilis phage recombinase, a Mycobacteriumtuberculosis phage recombinase and a Mycobacterium smegmatis phagerecombinase, provided that when the first and second recombinationattachment sites are attB, the third and fourth recombination attachmentsites are attP, and when the first and second recombination attachmentsites are attP, the third and fourth recombination attachment sites areattB. Preferably the recombinase is selected from the group consistingof an A118 recombinase, a SF370.1 recombinase, a SPβc2 recombinase, aφRv1 recombinase, and a Bxb1 recombinase.

Another embodiment of the present invention provides for a method forthe site-specific integration of a polynucleotide of interest into thegenome of a transgenic subject, wherein the genome comprises a firstrecombination attB or attP site or pseudo attB or pseudo attP site, themethod comprising: introducing a nucleic acid that comprises thepolynucleotide of interest and a second recombination attP or attB site;contacting the first and the second recombination sites with aprokaryotic recombinase polypeptide, wherein the recombinase polypeptidecan mediate site-specific recombination between the first and secondrecombination sites, and the recombinase is selected from the groupconsisting of a Listeria monocytogenes phage recombinase, aStreptococcus pyogenes phage recombinase, a Bacillus subtilis phagerecombinase, a Mycobacterium tuberculosis phage recombinase and aMycobacterium smegmatis phage recombinase, provided that when the firstrecombination site is attB or pseudo attB, the second recombination siteis attP and when the first recombination site is attP or pseudo attP,the second recombination site is attB. Preferably the recombinase isselected from the group consisting of an A118 recombinase, a SF370.1recombinase, a SPβc2 recombinase, a φRv1 recombinase, and a Bxb1recombinase.

Another method of the present invention provides for obtaining multiplesite-specific recombinations in a eukaryotic cell, the methodcomprising: providing a eukaryotic cell that comprises a firstrecombination site and a second recombination site with a thirdrecombination site and a fourth recombination site; contacting the firstand second recombination sites with a first prokaryotic recombinasepolypeptide, contacting the third and fourth recombination sites with asecond prokaryotic recombinase polypeptide, resulting in recombinationbetween the first and second recombination sites and recombinationbetween the third and fourth recombination sites, wherein the firstrecombinase polypeptide can mediate recombination between the first andsecond recombination sites and the second recombinase polypeptide canmediate recombination between the third and fourth recombination sites,the first and second recombinase are selected from the group consistingof a Listeria monocytogenes phage recombinase, a Streptococcus pyogenesphage recombinase, a Bacillus subtilis phage recombinase, aMycobacterium tuberculosis phage recombinase and a Mycobacteriumsmegmatis phage recombinase, provided that the first recombinasepolypeptide and the second recombinase polypeptide are different. Themethod can further comprising a fifth recombination site and a sixthrecombination site and a third recombinase polypeptide, wherein thethird recombinase polypeptide can mediate recombination between thefifth and sixth recombination sites, provided that the third recombinasepolypeptide is different than the first and second recombinasepolypeptides.

The present invention further relates to a eukaryotic cell thatcomprises a prokaryotic recombinase polypeptide or a nucleic acid thatencodes a prokaryotic recombinase, wherein the recombinase can mediatesite-specific recombination between a first recombination site and asecond recombination site that can serve as a substrate forrecombination with the first recombination site, wherein the firstrecombination site is attP, pseudo attP, attB or pseudo attB, the secondrecombination site is attB, pseudo attB, attP or pseudo attP, and therecombinase is selected from the group consisting of a Listeriamonocytogenes phage recombinase, a Streptococcus pyogenes phagerecombinase, a Bacillus subtilis phage recombinase, a Mycobacteriumtuberculosis phage recombinase and a Mycobacterium smegmatis phagerecombinase, provided that when the first recombination site is attB,the second recombination site is attP or pseudo attP, when the firstrecombination site is pseudo attB, the second recombination site isattP, when the first recombination site is attP, the secondrecombination site is attB or pseudo attB, and when the firstrecombination site is pseudo attP, the second recombination site isattB. Preferably the recombinase is selected from the group consistingof an A1188 recombinase, a SF370.1 recombinase, a SPβc2 recombinase, aφRv1 recombinase, and a Bxb1 recombinase.

Cells

Cells suitable for modification employing the methods of the inventioninclude both prokaryotic cells and eukaryotic cells. Prokaryotic cellsare cells that lack a defined nucleus. Examples of suitable prokaryoticcells include bacterial cells, mycoplasmal cells and archaebacterialcells. Particularly preferred prokaryotic cells include those that areuseful either in various types of test systems (discussed in greaterdetail below) or those that have some industrial utility such asKlebsiella oxytoca (ethanol production), Clostridium acetobulylicum(butanol production), and the like (see Green and Bennet, Biotech &Bioengineering 58:215-221, 1998; Ingram, et al, Biotech & Bioengineering58:204.206, 1998). Suitable eukaryotic cells include both animal cells(such as from insect, rodent, cow, goat, rabbit, sheep, non-humanprimate, human, and the like) and plant cells (such as rice, corn,cotton, tobacco, tomato, potato, and the like). Cell types applicable toparticular purposes are discussed in greater detail below.

Yet another embodiment of the invention comprises isolated geneticallyengineered cells. Suitable cells may be prokaryotic or eukaryotic, asdiscussed above. The genetically engineered cells of the invention maybe unicellular organisms or may be derived from multicellular organisms.By “isolated” in reference to genetically engineered cells derived frommulticellular organisms it is meant the cells are outside a living body,whether plant or animal, and in an artificial environment. The use ofthe term isolated does not imply that the genetically engineered cellsare the only cells present.

In one embodiment, the genetically engineered cells of the inventioncontain any one of die nucleic acid constructs of the invention. In asecond embodiment, a recombinase that specifically recognizesrecombination sequences is introduced into genetically engineered cellscontaining one of the nucleic acid constructs of the invention underconditions such that the nucleic acid sequence(s) of interest will beinserted into the genome. Thus, the genetically engineered cells possessa modified genome. Methods of introducing such a recombinase are wellknown in the art and are discussed above.

The genetically engineered cells of the invention can be employed in avariety of ways. Unicellular organisms can be modified to producecommercially valuable substances such as recombinant proteins,industrial solvents, industrially useful enzymes, and the like.Preferred unicellular organisms include fungi such as yeast (forexample, S. pombe, Pichia pastoris, S. cerevisiae (such as INVScl), andthe like) Aspergillis, and the like, and bacteria such as Klebsiella,Streptomyces, and the like.

Isolated cells from multicellular organisms can be similarly useful,including insect cells, mammalian cells and plant cells. Mammalian cellsthat may be useful include those derived from rodents, primates and thelike. They include Chinese Hamster Ovary (CHO) cells, HeLa cells, mouseneural stem cells, rat bone marrow stromal cells, cells of fibroblastorigin such as VERO, 3T3 or CHOK1, HEK 293 cells or cells of lymphoidorigin (such as 32D cells) and their derivatives.

In addition, plant cells, such as tobacco BY2 cells, are also availableas hosts, and control sequences compatible with plant cells areavailable, such as the cauliflower mosaic virus 35S and 19S, nopalinesynthase promoter and polyadenylation signal sequences, and the like.Appropriate transgenic plant cells can be used to produce transgenicplants.

Another preferred host is an insect cell, for example from theDrosophila larvae. Using insect cells as hosts, the Drosophila alcoholdehydrogenase promoter can be used (Rubin, Science 240:1453-1459, 1988).Alternatively, baculovirus vectors can be engineered to express largeamounts of peptide encoded by a desired nucleic acid sequence in insectcells (Jasny, Science 238:1653, 1987); Miller et al., In: GeneticEngineering (1986), Setlow, J. K., et al., eds., Plenum, Vol. 8, pp.277-297).

The genetically engineered cells of the invention are additionallyuseful as tools to screen for substances capable of modulating theactivity of a protein encoded by a nucleic acid fragment of interest.Thus, an additional embodiment of the invention comprises methods ofscreening comprising contacting genetically engineered cells of theinvention with a test substance and monitoring the cells for a change incell phenotype, cell proliferation, cell differentiation, enzymaticactivity of the protein or the interaction between the protein and anatural binding partner of the protein when compared to test cells notcontacted with the test substance.

A variety of test substances can be evaluated using the geneticallyengineered cells of the invention including peptides, proteins,antibodies, low molecular weight organic compounds, natural productsderived from, for example, fungal or plant cells, and the like. By “lowmolecular weight organic compound” it is, meant a chemical species witha molecular weight of generally less than 500-1000. Sources of testsubstances are well known to those of skill in the art.

Various assay methods employing cells are also well known by thoseskilled in the art. They include, for example, assays for enzymaticactivity (Hirth, et al, U.S. Pat. No. 5,763,198, issued Jun. 9, 1998),assays for binding of a test substance to a protein expressed by thegenetically engineered cells, assays for transcriptional activation of areporter gene, and the like.

Cells modified by the methods of the present invention can be maintainedunder conditions that, for example, (i) keep them alive but do notpromote growth, (ii) promote growth of the cells, and/or (iii) cause thecells to differentiate or dedifferentiate. Cell culture conditions aretypically permissive for the action of the recombinase in the cells,although regulation of the activity of the recombinase may also bemodulated by culture conditions (e.g., raising or lowering thetemperature at which the cells are cultured). For a given cell,cell-type, tissue, or organism, culture conditions are known in the art.

Transgenic Plants and Non-Human Animals

In another embodiment, the present invention comprises transgenic plantsand nonhuman transgenic animals whose genomes have been modified byemploying the methods and compositions of the invention. Transgenicanimals may be produced employing the methods of the present inventionto serve as a model system for the study of various disorders and forscreening of drugs that modulate such disorders.

A “transgenic” plant or animal refers to a genetically engineered plantor animal, or offspring of genetically engineered plants or animals. Atransgenic plant or animal usually contains material from at least oneunrelated organism, such as, from a virus. The term “animal” as used inthe context of transgenic organisms means all species except human. Italso includes an individual animal in all stages of development,including embryonic and fetal stages. Farm animals (e.g., chickens,pigs, goats, sheep, cows, horses, rabbits and the like), rodents (suchas mice), and domestic pets (e.g., cats and dogs) are included withinthe scope of the present invention. In a preferred embodiment, theanimal is a mouse or a rat.

The term “chimeric” plant or animal is used to refer to plants oranimals in which the heterologous gene is found, or in which theheterologous gene is expressed in some but not all cells of the plant oranimal.

The term transgenic animal also includes a germ cell line transgenicanimal. A “germ cell line transgenic animal” is a transgenic animal inwhich the genetic information provided by the invention method has beentaken up and incorporated into a germ line cell, therefore conferringthe ability to transfer the information to offspring. If such offspring,in fact, possess some or all of that information, then they, too, aretransgenic animals.

Methods of generating transgenic plants and animals are known in the artand can be used in combination with the teachings of the presentapplication.

In one embodiment, a transgenic animal of the present invention isproduced by introducing into a single cell embryo a nucleic acidconstruct, comprising a first recombination site capable of recombiningwith a second recombination site found within the genome of the organismfrom which the cell was derived and a nucleic acid fragment of interest,in a manner such that the nucleic acid fragment of interest is stablyintegrated into the DNA of germ line cells of the mature animal and isinherited in normal Mendelian fashion. In this embodiment, the nucleicacid fragment of interest can be any one of the fragment describedpreviously. Alternatively, the nucleic acid sequence of interest canencode an exogenous product that disrupts or interferes with expressionof an endogenously produced protein of interest, yielding a transgenicanimal with decreased expression of the protein of interest.

A variety of methods are available for the production of transgenicanimals. A nucleic acid construct of the invention can be injected intothe pronucleus, or cytoplasm, of a fertilized egg before fusion of themale and female pronuclei, or injected into the nucleus of an embryoniccell (e.g., the nucleus of a two-cell embryo) following the initiationof cell division (Brinster, et al., Proc. Nat. Acad. Sci. USA 82: 4438,1985). Embryos can be infected with viruses, especially retroviruses,modified with an attD recombination site and a nucleic acid sequence ofinterest. The cell can further be treated with a site-specificrecombinase as described above to promote integration of the nucleicacid sequence of interest into the genome.

By way of example only, to prepare a transgenic mouse, female mice areinduced to superovulate. After being allowed to mate, the females aresacrificed by CO₂ asphyxiation or cervical dislocation and embryos arerecovered from excised oviducts. Surrounding cumulus cells are removed.Pronuclear embryos are then washed and stored until the time ofinjection. Randomly cycling adult female mice are paired withvasectomized males. Recipient females are mated at the same time asdonor females. Embryos then are transferred surgically. The procedurefor generating transgenic rats is similar to that of mice. See Hammer,et al., Cell 63:1099-1112, 1990). Rodents suitable for transgenicexperiments can be obtained from standard commercial sources such asCharles River (Wilmington, Mass.), Taconic (Germantown, N.Y.), HarlanSprague Dawley (Indianapolis, Ind.), etc.

The procedures for manipulation of the rodent embryo and formicroinjection of DNA into the pronucleus of the zygote are well knownto those of ordinary skill in the art (Hogan, et al., supra).Microinjection procedures for fish, amphibian eggs and birds aredetailed in Houdebine and Chourrout, Experientia 47:897-905, 1991).Other procedures for introduction of DNA into tissues of animals aredescribed in U.S. Pat. No. 4,945,050 (Sandford et al., Jul. 30, 1990).

Totipotent or pluripotent stem cells derived from the inner cell mass ofthe embryo and stabilized in culture can be manipulated in culture toincorporate nucleic acid sequences employing invention methods. Atransgenic animal can be produced from such cells through injection intoa blastocyst that is then implanted into a foster mother and allowed tocome to term.

Methods for the culturing of stem cells and the subsequent production oftransgenic animals by the introduction of DNA into stem cells usingmethods such as electroporation, calcium phosphate/DNA precipitation,microinjection, liposome fusion, retroviral infection, and the like arealso are well known to those of ordinary skill in the art. See, forexample, Teratocarcinomas and Embryonic Stem Cells, A PracticalApproach, E. J. Robertson, ed., IRL Press, 1987). Reviews of standardlaboratory procedures for microinjection of heterologous DNAs intomammalian (mouse, pig, rabbit, sheep, goat, cow) fertilized ova include:Hogan et al., Manipulating the Mouse Embryo (Cold Spring Harbor Press1986); Krimpenfort et al., 1991, Bio/Technology 9:86: Palmiter et al.,1985, Cell 41:343; Kraemer et al., Genetic Manipulation of the EarlyMammalian Embryo (Cold Spring Harbor Laboratory Press 1985); Hammer etal., 1985, Nature, 315:680; Purcel et al., 1986, Science, 244:1281;Wagner et al., U.S. Pat. No. 5,175,385; Krimpenfort et al., U.S. Pat.No. 5,175,384, the respective contents of which are incorporated byreference.

The final phase of the procedure is to inject targeted ES cells intoblastocysts and to transfer the blastocysts into pseudo-pregnantfemales. The resulting chimeric animals are bred and the offspring areanalyzed by Southern blotting to identify individuals that carry thetransgene. Procedures for the production of non-rodent mammals and otheranimals have been discussed by others (see Houdebine and Chourrout,supra; Pursel, et al., Science 244:1281-1288, 1989; and Simms, et al.,Bio/Technology 6:179-183, 1988). Animals carrying the transgene can beidentified by methods well known in the art, e.g., by dot blotting orSouthern blotting.

The term transgenic as used herein additionally includes any organismwhose genome has been altered by in vitro manipulation of the earlyembryo or fertilized egg or by any transgenic technology to induce aspecific gene knockout. The term “gene knockout” as used herein, refersto the targeted disruption of a gene in vivo with loss of function thathas been achieved by use of the invention vector. In one embodiment,transgenic animals having gene knockouts are those in which the targetgene has been rendered nonfunctional by an insertion targeted to thegene to be rendered non-functional by targeting a pseudo-recombinationsite located within the gene sequence.

Gene Therapy and Disorders

A further embodiment of the invention comprises a method of treating adisorder in a subject in need of such treatment. In one embodiment ofthe method, at least one cell or cell type (or tissue, etc.) of thesubject has a recombination site. This cell(s) is transformed with anucleic acid construct (a “targeting construct”) comprising a secondrecombination sequence and one or more polynucleotides of interest(typically a therapeutic gene). into the same cell a recombinase isintroduced that specifically recognizes the recombination sequencesunder conditions such that the nucleic acid sequence of interest isinserted into the genome via a recombination event between the first andsecond recombination sites. Subjects treatable using the methods of theinvention include both humans and non-human animals. Such methodsutilize the targeting constructs and recombinases of the presentinvention.

A variety of disorders may be treated by employing the method of theinvention including monogenic disorders, infectious diseases, acquireddisorders, cancer, and the like. Exemplary monogenic disorders includeADA deficiency, cystic fibrosis, familial-hypercholesterolemia,hemophilia, chronic ganulomatous disease, Duchenne muscular dystrophy,Fanconi anemia, sickle-cell anemia, Gaucher's disease, Hunter syndrome,X-linked SCID, and the like.

Infectious diseases treatable by employing the methods of the inventioninclude infection with various types of virus including human T-celllymphotropic virus, influenza virus, papilloma virus, hepatitis virus,herpes virus, Epstein-Bar virus, immunodeficiency viruses (HIV, and thelike), cytomegalovirus, and the like. Also included are infections withother pathogenic organisms such as Mycobacterium Tuberculosis,Mycoplasma pneumoniae, and the like or parasites such as Plasmadiumfalciparum, and the like.

The term “acquired disorder” as used herein refers to a noncongenitaldisorder. Such disorders are generally considered more complex thanmonogenic disorders and may result from inappropriate or unwantedactivity of one or more genes. Examples of such disorders includeperipheral artery disease, rheumatoid arthritis, coronary arterydisease, and the like.

A particular group of acquired disorders treatable by employing themethods of the invention include various cancers, including both solidtumors and hematopoietic cancers such as leukemias and lymphomas. Solidtumors that are treatable utilizing the invention method includecarcinomas, sarcomas, osteomas, fibrosarcomas, chondrosarcomas, and thelike. Specific cancers include breast cancer, brain cancer, lung cancer(non-small cell and small cell), colon cancer, pancreatic cancer,prostate cancer, gastric cancer, bladder cancer, kidney cancer, head andneck cancer, and the like.

The suitability of the particular place in the genome is dependent inpart on the particular disorder being treated. For example, if thedisorder is a monogenic disorder and the desired treatment is theaddition of a therapeutic nucleic acid encoding a non-mutated form ofthe nucleic acid thought to be the causative agent of the disorder, asuitable place may be a region of the genome that does not encode anyknown protein and which allows for a reasonable expression level of theadded nucleic acid. Methods of identifying suitable places in the genomeare well known in the art and described further in the Examples below.

The nucleic acid construct useful in this embodiment is additionallycomprised of one or more nucleic acid fragments of interest. Preferrednucleic acid fragments of interest for use in this embodiment aretherapeutic genes and/or control regions, as previously defined. Thechoice of nucleic acid sequence will depend on the nature of thedisorder to be treated. For example, a nucleic acid construct intendedto treat hemophilia B, which is caused by a deficiency of coagulationfactor IX, may comprise a nucleic acid fragment encoding functionalfactor IX. A nucleic acid construct intended to treat obstructiveperipheral artery disease may comprise nucleic acid fragments encodingproteins that stimulate the growth of new blood vessels, such as, forexample, vascular endothelial growth factor, platelet-derived growthfactor, and the like. Those of skill in the art would readily recognizewhich nucleic acid fragments of interest would be useful in thetreatment of a particular disorder.

The nucleic acid construct can be administered to the subject beingtreated using a variety of methods. Administration can take place invivo or c vivo. By “in vivo,” it is meant in the living body of ananimal. By “ex vivo” it is meant that cells or organs are modifiedoutside of the body, such cells or organs are typically returned to aliving body.

Methods for the therapeutic administration of nucleic acid constructsare well known in the art. Nucleic acid constructs can be delivered withcationic lipids (Goddard, et al, Gene Therapy, 4:1231-1236, 1997;Gorman, et al, Gene Therapy 4:983-992, 1997; Chadwick, et al, GeneTherapy 4:937-942, 1997, Gokhale, et al, Gene Therapy 4:1289-1299, 1997;Gao, and Huang, Gene Therapy 2:710-722, 1995, all of which areincorporated by reference herein), using viral vectors (Monahan, et al,Gene Therapy 4:40-49, 1997; Onodera, et al, Blood 91:30-36, 1998, all ofwhich are incorporated by reference herein), by uptake of “naked DNA”,and the like. Techniques well known in the art for the transfection ofcells (see discussion above) can be used for the ex vivo administrationof nucleic acid constructs. The exact formulation, route ofadministration and dosage can be chosen by the individual physician inview of the patient's condition. (See e.g. Fingl et al., 1975, in “ThePharmacological Basis of Therapeutics”, Ch. 1 p. 1).

It should be noted that the attending physician would know how to andwhen to terminate, interrupt, or adjust administration due to toxicity,to organ dysfunction, and the like. Conversely, the attending physicianwould also know how to adjust treatment to higher levels if the clinicalresponse were not adequate (precluding toxicity). The magnitude of anadministered dose in the management of the disorder being treated willvary with the severity of the condition to be treated, with the route ofadministration, and the like. The severity of the condition may, forexample, be evaluated, in part, by standard prognostic evaluationmethods. Further, the dose and perhaps dose frequency will also varyaccording to the age, body weight, and response of the individualpatient.

In general at least 1-10% of the cells targeted for genomic modificationshould be modified in the treatment of a disorder. Thus, the method androute of administration will optimally be chosen to modify at least0.1-1% of the target cells per administration. In this way, the numberof administrations can be held to a minimum in order to increase theefficiency and convenience of the treatment.

Depending on the specific conditions being treated, such agents may beformulated and administered systemically or locally. Techniques forformulation and administration may be found in “Remington'sPharmaceutical Sciences,” 1990, 18th ed., Mack Publishing Co., Easton,Pa. Suitable routes may include oral, rectal, transdermal, vaginal,transmucosal, or intestinal administration; parenteral delivery,including intramuscular, subcutaneous, intramedullary injections, aswell as intrathecal, direct intraventricular, intravenous,intraperitoneal, intranasal, or intraocular injections, just to name afew.

The subject being treated will additionally be administered arecombinase that specifically recognizes the first and secondrecombination sequences that are selected for use. The particularrecombinase can be administered by including a nucleic acid encoding itas part of a nucleic acid construct, or as a protein to be taken up bythe cells whose genome is to be modified. Methods and routes ofadministration will be similar to those described above foradministration of a targeting construct comprising a recombinationsequence and nucleic acid sequence of interest. The recombinase proteinis likely to only be required for a limited period of time forintegration of the nucleic acid sequence of interest. Therefore, ifintroduced as a recombinase gene, the vector carrying the recombinasegene will lack sequences mediating prolonged retention. For example,conventional plasmid DNA decays rapidly in most mammalian cells. Therecombinase gene may also be equipped with gene expression sequencesthat limit its expression. For example, an inducible promoter can beused, so that recombinase expression can be temporally regulated bylimited exposure to the inducing agent. One such exemplary group ofpromoters is ecdysone-responsive promoters, the expression of which canbe regulated using ecdysteroids or other non-steroidal agonists. Anothergroup of promoters are tetracycline-responsive promoters, the expressionof which can be regulated using tetracycline or doxycycline.

EXAMPLES General Methods

Standard recombinant DNA and molecular cloning techniques used hereinare well known in the att and are described by Sambrook, J., Fritsch, E.F. and Maniatis, T. Molecular Cloning: A Laboratory Manual; Cold SpringHarbor Laboratory Press: Cold Spring Harbor, N.Y. (1989) and by T. J.Silhavy, M. L. Bennan, and L. W. Enquist, Experiments with Gene Fusions,Cold Spring Harbor Laboratory. Cold Spring Harbor, N.Y. (1984), and byAusubel, F. M. et al., Current Protocols in Molecular Biology, GreenePublishing Assoc. and Wiley-Interscience. New York, N.Y. (1987).Materials and methods suitable for the maintenance and growth ofbacterial cultures are well known in the art. Techniques suitable foruse in the following examples may be found as set out in Phillipp, G. etal., Manual of Methods fir General Bacteriology, American Society forMicrobiology, Washington, D.C. (1994) or in Brock, T. D. Biotechnology:A Textbook of Industrial Microbiology, Second Edition, SinauerAssociates, Inc., Sunderland, Mass. (1989). All reagents, restrictionenzymes and materials used for the growth and maintenance of host cellswere obtained from New England Biolabs (Beverly, Mass.), InvitrogenCorporation (Carlsbad, Calif.), Stratagene Corporation (La Jolla,Calif.), Promega Corporation (Madison, Wis.), DIFCO Laboratories(Detroit, Mich.), or Sigma/Aldrich Chemical Company (St. Louis, Mo.)unless otherwise specified.

Manipulations of genetic sequences and alignment and comparison ofpolynucleotide and peptide sequences can be accomplished using the suiteof programs available from Invitrogen Corporation, Carlsbad, Calif.(Vector NTI software version 8.0), DNASTAR, Inc., Madison, Wis. (DNASTARsoftware version 6.0), or Genetics Computer Group Inc., Madison, Wis.(Wisconsin Package Version 9.0).

The meaning of abbreviations is as follows: “h” means hour(s), “μL”means microliter(s), “mL” means milliliter(s), “L” means liter(s), “μM”means micromolar, “mM” means millimolar, “ng” means nanogram(s), “μg”means microgram(s), “mg” means milligram(s), “A” means adenine oradenosine, “T” means thymine or thymidine, “G” means guanine orguanosine, “C” means cytidine or cytosine, “nt” means nucleotide(s),“aa” means amino acid(s), “bp” means base pair(s), “kb” meanskilobase(s), “k” means kilo, “μL” means micro, “φ” means Phi, “β” meansbeta. “SE” means standard error, “Luc” means firefly luciferase, “RLuc”means Renilla luciferase, and “° C.” means degrees Celsius.

The following examples demonstrate that site-specific recombinasesystems derived from Bacillus subtilis bacteriophage SPβc2,Streptococcus pyogenes bacteriophage SF370.1, Mycobacterium smegmatisbacteriophage Bxb1, Listeria monocytogenes bacteriophage A118, andMycobacterium tuberculosis bacteriophage φRv1 function in eukaryoticcells.

These examples are offered to illustrate, but not to limit the presentinvention.

Example 1 Design, Synthesis and Cloning of Recombinase Genes andIntramolecular Recombination Assay Plasmids

After analyzing the published literature and sequences available inGenbank, numerous site-specific recombinases were selected and assayedfor DNA integration, excision, inversion, and replacement in mammalianand plant cells. The amino acid sequences for large site-specificrecombinases of serine family (Smith, M. C. and H. M. Thorpe 2000Diversity in the serine recombinases. Mol. Microbiol., 44:299-307) wereobtained from GenBank and reverse translated to DNA. Since the sourcesof recombinases were from bacteria or bacterial viruses, we optimizedthe DNA sequence for recombinase expression in mammalian cells withoutchanging the encoded amino acid sequence. The genes were totallysynthesized using the codons for high-level human and mouse expressionand with convenient restriction enzyme sites for cloning. In addition,regions of very high (>80%) or very low (<30%) GC content have beenavoided where possible. Moreover, during the optimization the followingcis-acting sequence motifs were avoided to optimize RNA stability andtranslation:

-   -   internal TATA-boxes, chi-sites and ribosomal entry sites    -   AT-rich or GC-rich sequence stretches    -   repeat sequences and RNA secondary structures    -   (cryptic) splice donor and acceptor sites, branch points    -   poly(A) sites        The codon and RNA optimization resulted in difference of 20-30%        of sequence between native (i.e., DNA sequence available at        Genbank) and synthetic genes. The synthetic genes encoding the        recombinases were cloned into mammalian and E. coli expression        plasmid pDual obtained from Stratagene Corporation (La Jolla,        Calif., catalog #214501). pDual expression vector directs        expression of heterologous genes in both mammalian and        prokaryotic cells. For the constitutive expression in mammalian        cells the vector contains the promoter/enhancer of the human        cytomegalovirus (CMV) immediate early gene. The recombinase gene        is cloned at the unique Eam 1104 I restriction enzyme site        present between the CMV promoter and SV40 terminator sequence.        While synthesizing the gene sequences we added Eam 1104 I        restriction enzyme recognition site at the beginning (before the        initiation codon ATG) and end (after the stop codon TAG) of the        gene to facilitate digestion with Eam 1104 I enzyme and cloning        at the same site in the pDual plasmid. The cloning of synthetic        genes, sequencing of clones to confirm the gene sequence after        cloning into pDual vector were performed using the standard DNA        cloning procedures (Sambrook, J., E. F. Fritsch, et al. 1989.        Molecular Cloning: A laboratory Manual. Cold Spring Harbor        Press, Cold Spring Harbor, N.Y.). The description of expression        plasmids is given below.

1.1 SPβc2 Recombinase Expression Plasmid:

A synthetic DNA sequence (SEQ ID NO: 1) codon optimized for animal cellexpression and encoding the site-specific DNA recombinase yokA ofBacillus subtilis phage SPβc2 (SEQ ID NO: 2, Genbank accession #T12765,Lazarevic, V., A. Dusterhoft, et al. 1999, Nucleotide sequence of theBacillus subtilis temperate bacteriophage SPβc2. Microbiology145:1055-67) was cloned into pDual expression vector at Eam 1104 Irestriction site following the procedures recommended by Stratagene (LaJolla, Calif.).

1.2 SF370.1 Recombinase Expression Plasmid:

A synthetic DNA sequence (SEQ ID NO: 3) codon optimized for animal cellexpression and encoding the putative recombinase of Streptococcuspyogenes bacteriophage SF370.1 (SEQ ID NO: 4, Genbank accession #T12765,Canchaya, C., F. Desiere, et al. 2002, Genome analysis of an inducibleprophage and prophage remnants integrated in the Streptococcus pyogenesstrain SF370. Virology 302:245-58) was cloned into pDual expressionvector at Eam 1104 I restriction site following the proceduresrecommended by Stratagene (La Jolla, Calif.).

1.3 Bxb1 Recombinase Expression Plasmid:

A synthetic DNA sequence (SEQ ID) NO: 5) codon optimized for animal cellexpression and encoding the putative recombinase of Mycobacteriumsmegmatis bacteriophage Bxb1 (SEQ ID NO: 6, Genbank accession #AAG59740,Mediavilla, J., S. Jain, et al. 2000, Genome organization andcharacterization of mycobacteriophage Bxb1. Mol. Microbiol. 38:955-70)was cloned into pDual expression vector at Eam 1104 I restriction sitefollowing the procedures recommended by Stratagene (La Jolla, Calif.).1.4 a 118 Recombinase Expression Plasmid:

A synthetic DNA sequence (SEQ ID NO: 7) codon optimized for animal cellexpression and encoding the putative recombinase of Listeriamonocytogenes bacteriophage A118 (SEQ ID NO: 8, Genbank accession#CAB53817, Loessner, M. J., R. B. Inman, et al. 2000, Completenucleotide sequence, molecular analysis and genome structure ofbacteriophage A118 of Listeria monocytogenes: implications for phageevolution. Mol. Microbiol. 35:324-40) was cloned into pDual expressionvector at Eam 1104 I restriction site following the proceduresrecommended by Stratagene (La Jolla, Calif.).

1.5 φRv1 Recombinase Expression Plasmid:

A synthetic DNA sequence (SEQ ID NO: 9) codon optimized for animal cellexpression and encoding the putative recombinase of and Mycobacteriumtuberculosis bacteriophage φRv1 (SEQ ID NO: 10, Genbank accession#CAB09083, Bibb, L. A. and G. F. Hatful 12002, Integration and excisionof the Mycobacterium tuberculosis prophage-like element, phiRv1. Mol.Microbiol. 45:1515-26) was cloned into pDual expression vector at Eam1104 I restriction site following the procedures recommended byStratagene (La Jolla, Calif.).

1.6 A118 Recombinase Plant Expression Plasmid:

A synthetic DNA sequence (SEQ ID NO: 7) codon optimized for animal cellexpression and encoding the putative recombinase of Listeriamonocytogenes bacteriophages A118 was cloned into plant expressionplasmid pILTAB358 between the cassava vein mosaic promoter NOSterminator sequence (Verdaguer, B., A. Kochko et al. 1998, Functionalorganization of the cassava vein mosaic virus (CsVMV) promoter. PlantMol. Biol. 37:1055-67). pILTAB plasmid DNA was obtained from DonaldDanforth Center for Plant Research, St. Louis, Mo. The constructs aresimilar to the A118 expression plasmid used in animal cells except thatthe CMV promoter and SV40 terminator were replaced with cassava veinmosaic promoter and 35S terminator, respectively.

Design and Construction of Intramolecular Recombination Assay Plasmids

Intramolecular recombination assay plasmids were constructed using theplasmid gWiz™ Luc (Gene Therapy Systems, San Diego, Calif.). Thisplasmid confers kanamycin resistance in E coil and expresses aluciferase gene constitutively from the CMV promoter when introducedinto mammalian cells. The vector also contains unique Sal I and Not Irestriction sites between the CMV promoter and start codon of luciferasegene. Recognition sites for restriction enzymes Apa L and Nhe I werecreated by inserting an oligonucleotide between the Sal I and Not Isites. Oligonucleotides containing the attP site of recombinase andhaving Sal I and Apa I flanking restriction sites were synthesized,annealed, and inserted between the Sal and Apa I sites. Similarly,oligonucleotides containing the attB sequence were inserted between theNhe I and Not I sites. A 1296 bp transcriptional termination or STOPsequence was PCR amplified from plasmid pBS302 (Genbank accession#U51223, nucleotides 193-1488) and cloned at Apa I and Nhe I sites,between attP and attB sites. The final construct had the attP. STOP, andattB sequences placed between the CMV promoter and luciferase gene asshown in FIG. 1. The plasmid would express luciferase gene only afterthe deletion of STOP sequence due to recombination between attP and attBsites. The description of intramolecular recombination assay plasmids isgiven below.

1.7 SPβc2 Intramolecular Recombination Assay Plasmid:

A 99 bp synthetic oligonucleotide sequence containing the attP site ofSPβc2 recombinase (SEQ ID NO: 11), a 1296 bp STOP sequence (SEQ ID NO:12), and a 96 bp synthetic oligonucleotide sequence containing the attBsite (SEQ ID NO: 13) of SPβc2 recombinase were cloned in that orderbetween the CMV promoter and luciferase gene of gWiz™ Luc plasmid.

1.8 SF370.1 Intramolecular Recombination Assay Plasmid:

A 99 bp synthetic oligonucleotide sequence containing the attP site ofSF370.1 recombinase (SEQ ID NO: 14), a 1296 bp STOP sequence (SEQ ID NO:12), and a 96 bp synthetic oligonucleotide sequence containing the attBsite (SEQ ID NO: 15) of SF370.1 recombinase were cloned in that orderbetween the CMV promoter and luciferase gene of gWiz™ Luc plasmid.

1.9 Bxb1 Intramolecular Recombination Assay Plasmid:

A 52 bp synthetic oligonucleotide sequence containing the attP site ofBxb1 recombinase (SEQ ID NO: 16), a 1296 bp STOP sequence (SEQ ID NO:12), and a 46 bp synthetic oligonucleotide sequence containing the attBsite (SEQ ID NO: 17) of Bxb1 recombinase were cloned in that orderbetween the CMV promoter and luciferase gene of gWiz™ Luc plasmid.

1.10 A118 Intramolecular Recombination Assay Plasmid:

A 99 bp synthetic oligonucleotide sequence containing the attP site ofA118 recombinase (SEQ ID NO: 18), a 1296 bp STOP sequence (SEQ ID NO:12), and a 96 bp synthetic oligonucleotide sequence containing the attBsite (SEQ ID NO: 19) of A118 recombinase were cloned in that orderbetween the CMV promoter and luciferase gene of gWiz™ Luc plasmid.

1.11 φRv1 Intramolecular Recombination Assay Plasmid:

A 99 bp synthetic oligonucleotide sequence containing the attP site ofφRv1 recombinase (SEQ ID NO: 20), a 1296 bp STOP sequence (SEQ ID NO:12), and a 96 bp synthetic oligonucleotide sequence containing the attBsite (SEQ ID NO: 21) of φRv1 recombinase were cloned in that orderbetween the CMV promoter and luciferase gene of gWiz™ Luc plasmid.

1.12 A118 Intramolecular Recombination Assay Plant Plasmid:

A 99 bp synthetic oligonucleotide sequence containing the attP site ofA118 recombinase (SEQ ID NO: 18), a 1296 bp STOP sequence (SEQ ID NO:12), a 96 bp synthetic oligonucleotide sequence containing the attB site(SEQ ID NO: 19) of A118 recombinase, and luciferase gene were cloned inthat order between the cassava vein mosaic promoter and NOS terminatorsequence of pILTAB358.

Example 2 Transient Intramolecular Recombination Assays

In order to determine the activity of the recombinases in mammalian andplant cells, a transient assay was developed. Briefly, the assayconsisted of cloning the recombinase gene into an expression plasmid,making the corresponding intramolecular recombination assay plasmid,introducing both plasmid DNAs into cells by transfection, and assayingfor luciferase enzyme activity. The recombinase assay plasmids containedCMV Promoter-attP:STOP:attB—Luciferase Reporter gene—Terminatorsequences. The STOP sequence is a transcription termination signalsequence. In the absence of recombination, expression of the luciferasereporter gene is prevented by the STOP sequence present between thepromoter and reporter gene. Recombination between the attP and attBsites due to the introduced recombinase results in deletion of the STOPsequence and activation of reporter gene. This assay is sensitive androbust because it is an OFF to ON format and the amount of luciferasereporter can be easily assayed by detecting the light emitted byluciferase with a luminometer. The assay format is graphically depictedin FIG. 1.

Transient Transfections and Luciferase Assays

Cells were maintained at 37° C. and 5% CO₂ in DMEM supplemented with 10%fetal bovine serum and 1% penicillin/streptomycin (obtained fromInvitrogen, Carlsbad, Calif.) or in other media as indicated. On the dayof transfection, cells were plated at different densities depending onthe cell type used. The cells were transfected with intramolecularrecombination assay plasmid alone or along with varying amounts ofrecombinase expression plasmid DNA using Lipofectamine 2000™ accordingto the manufacturers instructions (Invitrogen, Carlsbad, Calif.).Constitutively expressed Renilla luciferase reporter plasmid (pRL-CMVfrom Promega, Madison, Wis.) was co-transfected (2 ng/well) and used asan internal control to normalize the data. Twenty-four or forty-eighthours after transfection (depending on the cell line), media wasdiscarded and cells were lysed with passive lysis buffer (Promega,Madison, Wis.). Extracts were then assayed using Dual Luciferase Assaykit (Promega, Madison, Wis.) on a plate reader equipped with injectors(Dynex Technologies, Chantilly, Va.). The data shown are the ratios ofluciferase (Luc) and Renilla luciferase (RLuc) activities, unless notedotherwise. Similar results were observed when Luc activities (relativelight units) were compared (data not shown). Since the number ofreplicates and experiments varied for different constructs and celllines the standard error was used to indicate the experimentalvariation.

2.1 Transient Intramolecular Recombination Assay in Human HEK293 Cells

Cells (20,000 cells per well in a 96-well plate) were transfected with25 ng of intramolecular recombination assay plasmid and 0, 10, 25, or 75ng of the corresponding recombinase plasmid and incubated for 24 hours.Cells were lysed with 50 μl of passive lysis buffer and 25 μl extractswere assayed. Six to twenty replicate assays were performed, and ratiosof Luc/RLuc (mean values)±SE were plotted. The values shown above thebars in FIG. 2 are fold inductions (ratio of luciferase activity in thepresence of recombinase plasmid to the activity in the absence ofrecombinase plasmid).

As shown in FIG. 2, transfection of intramolecular recombination assayplasmid alone showed no or very little luciferase activity (given asratio of Luc/RLuc). Transfection of increasing amounts of A118recombinase expression plasmid (10, 25, or 75 ng) along with A118intramolecular recombination assay plasmid increased the luciferaseactivity. Similar results were also observed for SF370.1, SPβc2, φRV1,and Bxb1. These results clearly indicated that the recombinases arefunctional in HEK293 cells. The recombinases mediated the recombinationbetween their attP and attB sites and deleted the STOP sequence on theintramolecular recombination assay plasmid and activated the luciferasegene expression.

2.2 Transient Intramolecular Recombination Assay in Mouse NIH3T3 Cells

Cells (5,000 cells per well in a 96-well plate) were transfected with 25ng of intramolecular recombination assay plasmid and 0, 10, 25, or 75 ngof the corresponding recombinase expression plasmid and incubated for 24hours. Cells were lysed with 50 μl of passive lysis buffer and 25 μlextracts were assayed. Two to fourteen replicate assays were performed,and ratios of Luc/RLuc (mean values) SE were plotted. The values shownabove the bars in FIG. 3 are fold inductions.

FIG. 3 shows the data obtained from transfection of NIH3T3 withintramolecular recombination assay plasmid alone or along withincreasing amounts (10, 25, or 75 ng) of recombinase expression plasmid.Co-transfection of recombinase plasmid and intramolecular recombinationassay plasmid increased the luciferase activity many fold. For example,transfection of cells with 25 ng Bxb1 intramolecular recombination assayplasmid and 75 ng of Bxb1 recombinase expression plasmid increased theluciferase activity 66-fold when compared with transfection with 25 ngBxb1 intramolecular recombination assay plasmid alone. Similar to Bxb1,recombinases A118, SF370.1, SPβc2, and φRV1 also increased theluciferase activity (FIG. 3) showing that these recombinases arefunctional in mouse NIH3T3 cells and are effective at recombining theirattP and attB sites.

2.3 Transient Intramolecular Recombination Assay in Chinese HamsterOvary (CHO) Cells

Cells (15,000 cells per well in a 96-well plate) were transfected with25 ng of intramolecular recombination assay plasmid and 0, 10, 25, or 75ng of the corresponding recombinase expression plasmid and incubated for24 hours. Cells were lysed with 50 μl of passive lysis buffer and 25 μlextracts were assayed. Two to eight replicate assays were performed, andratios of Luc/RLuc (mean values)±SE were plotted. The values shown abovethe bars in FIG. 4 are fold inductions.

As shown in FIG. 4, transfection of intramolecular recombination assayplasmid of A118, SF370.1, or φRV1 alone showed no or very littleluciferase activity. Co-transfection with increasing amounts ofcorresponding A118, SF370.1, or φRV1 recombinase expression plasmidincreased the luciferase activity. These results clearly indicated thatthe recombinases are functional in CHO cells. The recombinases mediatedthe recombination between their attP and attB sites and deleted the STOPsequence on the intramolecular recombination assay plasmid and activatedthe luciferase gene expression.

2.4 Transient Intramolecular Recombination Assay in Human HeLa Cells

Cells (15,000 cells per well in a 96-well plate) were transfected with25 ng of intramolecular recombination assay plasmid and 0, 10, 25, or 75ng of the corresponding recombinase expression plasmid and incubated for24 hours. Two to eight replicate assays were performed, and ratios ofLuc/RLuc (mean values)±SE were plotted. The values shown above the barsin FIG. 5 are fold inductions.

As shown in FIG. 5, transfection of intramolecular recombination assayplasmid of A118, SF370.1, or φRV1 alone showed no or very littleluciferase activity. Co-transfection with increasing amounts ofcorresponding A118, SF370.1, or φRV1 recombinase expression plasmidincreased the luciferase activity. These results showed that therecombinases are functional in HeLa cells.

2.5 Transient Intramolecular Recombination Assay in Rat Bone MarrowStromal Cells

Primary bone marrow stromal cells from rats were pre-plated one daybefore the transfection at a density of 4000 cells/cm² and cultured inmedium containing 50% Minimum Essential Medium Alpha Medium (αMEM), 50%F12 Hams, 10% FBS, 1% Pen/Strep (100 U/ml penicillin G and 100 mg/mlstreptomycin sulfate). Cells were transfected with 25 ng ofintramolecular recombination assay plasmid and 0, 50, 100, or 200 ng ofthe corresponding recombinase plasmid and incubated for 48 hours. Cellswere lysed with 50 μl of passive lysis buffer and 25 μl extracts wereassayed. Eight replicate assays were performed, and ratios of Luc/RLuc(mean values). SE were plotted. The values shown above the bars in FIG.6 are fold inductions.

FIG. 6 shows the data obtained from transfection of rat bone marrowstromal cells with intramolecular recombination assay plasmid alone oralong with increasing amounts (50, 100, or 200 ng) of correspondingrecombinase expression plasmid. Co-transfection of intramolecularrecombination assay plasmid and recombinase expression plasmid increasedthe luciferase activity many fold. For example, transfection of cellswith 25 ng Bxb1 intramolecular recombination assay plasmid and 200 ng ofBxb1 recombinase expression plasmid increased the luciferase activity501-fold when compared to transfection with 25 ng Bxb1 intramolecularrecombination assay plasmid alone. Similar to Bxb1, recombinases A118,SF370.1, SPβc2, and +RV also increased the luciferase activity (FIG. 6)showing that these recombinases are functional in rat bone marrowstromal cells and are effective at recombining their attP and attBsites.

2.6 Transient Intramolecular Recombination Assay in Mouse Neural StemCells

Mouse neural stem C17.2 cells (mNSCs) were obtained from Dr. Evan Snyderof The Burnham Research Institute, La Jolla, Calif. and maintained usingthe recommended protocol (Ryder, E. F., E. Y. Snyder, et al. 1990.Establishment and characterization of multipotent neural cell linesusing retrovirus vector-mediated oncogene transfer. J. Neurobiol.,21:356-75). Cells were split one day prior to transfection and plated in48-well plates at a density of 120,000 cells per well. After overnightincubation the culture media was replaced with serum-free medium. Thecells were transfected with 50 ng intramolecular recombination assayplasmid alone or along with 0, 25, 50, 100, or 200 ng of recombinaseplasmid DNA using transfection reagent Lipofectamine 2000™ according tothe manufacturers instructions (Invitrogen, Carlsbad, Calif.).Constitutively expressed Renilla luciferase reporter plasmid (pRL-CMV,Promega, Madison, Wis.) was co-transfected (4 ng/well) as an internalcontrol to normalize the data. Two days after transfection, the mediawas discarded and cells were lysed with 75 μl of passive lysis buffer(Promega, Madison, Wis.). Extracts (50 μl) were assayed for luciferaseand Renilla luciferase activities using the Dual Luciferase Assay kit(Promega, Madison, Wis.) on a plate reader equipped with injectors(Dynex Technologies, Chantilly, Va.). The data shown in FIG. 7 are theratios of luciferase (Luc) and Renilla luciferase (RLuc) activities, andis the average of 4 transfections per treatment. Error bars representstandard error.

Similar to results observed in HEK293, NIH3T3; CHO, HeLa, and rat bonemarrow stromal cells, recombinases A118, SF370.1, SPβc2, φRV1, and Bxb1were functional in mNSCs and increased the luciferase activity (FIG. 7).Co-transfection of increasing amounts (25, 50, 100, or 200 ng) ofrecombinase expression plasmid with corresponding intramolecularrecombination assay plasmid (50 ng) resulted in higher luciferaseactivities and the fold inductions ranged from 72-5349.

2.7 Transient Intramolecular Recombination Assay in Tobacco BY2 Cells

Cell suspension cultures of Nicotiana tobacum BY2 were maintained in MSmedium in the dark and subcultured weekly (Nagata, T., T. Nemoto, and S.Hasezawa. 1992. Tobacco BY-2 cell line as the Hela cell in the cellbiology of higher plants. Intl. Rev. Cytol., 132:1-30). Protoplastsprepared from 3 day-old cultures were resuspended in 0.4 M mannitol anddistributed into 35 mm petri dishes in 1 mL aliquots (˜5×10⁵ cells).Protoplasts were mixed with plasmid DNA and electroporated at 0.56 KVolts for 80 It seconds using a square wave electroporation system withPetripulser electrode (BTX, San Diego, Calif., USA). The cells weretransfected with 10 μg for the intramolecular recombination test plasmidand 0 or 10 f.!g for the recombinase expression plasmid. Following theelectroporation, protoplasts were diluted with 1 mL of 2× protoplastculture medium (Watanabe, Y., T. Meshi, and Y. Okada. 1987. Infection oftobacco protoplasts with in vitro transcribed tobacco mosaic virus RNAusing an improved electroporation method. Virology, 192:264-272),aliquotted as two 1 mL cultures, and incubated at 27° C. for 17 h.Protoplasts were lysed by freeze thawing and addition of 250 μL 5×passive lysis buffer (Promega, Madison, Wis., USA). Twenty μL of cellextract was assayed for luciferase activity using Dual Luciferase Assaykit on a plate reader equipped with injectors. The data shown in FIG. 8are the relative light units due to luciferase activity. The valuesshown are average of 22 replicates and the error bars are standarderror.

As shown in FIG. 8, transfection of BY2 cells with A118 intramolecularrecombination plant assay plasmid alone showed very little luciferaseactivity. Co-transfection with A118 recombinase plant expression plasmidresulted in 364-fold increase in luciferase activity. The data clearlyindicated that the recombinase recombined attP and attB sites in plantcells.

Example 3 Stable Integration of Plasmid DNA Containing attP or attBSequence into HEK293 Chromosome Containing the attB or attP Site

Assay for the integration of plasmid DNA at attP or attB site on thechromosome was done in a two-step process. In the first step, a stablecell line containing a single copy of attP or attB site of each enzymewas generated and characterized. In the second step, a plasmidcontaining the attP or attB site was integrated at the chromosomal attBor attP, respectively, in the presence of the recombinase expressionplasmid.

Generation of Stable HEK293 Clones with attP or attB Sequence in theChromosome

A single copy of attP or attB sequence of each recombinase (SEQ IDNumbers 11, 13-21) was introduced at the FRT locus in Flp-In™-293 cellsobtained from Invitrogen [Carlsbad, Calif. (catalog #R750-07)] followingthe procedure recommended by the manufacturer. The FRT locus inFlp-In™-293 cells has a CMV promoter, FRT integration site for Flprecombinase, and zeocin resistance and β-galactosidase fusion gene.These cells grow in the presence of zeocin antibiotic and expressβ-galactosidase marker gene. The attP or attB sequence of each enzymewas cloned into pcDNA/FRT plasmid (Invitrogen, Carlsbad, Calif., catalog#V6010-20) at the multiple cloning sites region present between the CMVpromoter and BGH terminator sequence. The pcDNA/FRT cloning plasmid hasa FRT site preceding the hygromycin gene. The hygromycin gene lacks apromoter and ATG initiation codon. Therefore, transfection of pcDNA/FRTplasmid containing the attP or attB site into mammalian cells will notconfer hygromycin resistance. The integration of pcDNA/FRT plasmidoccurs at the FRT locus in Flp-In™-293 cells only followingco-transfection with the Flp recombinase expression plasmid (pCG44,Invitrogen, Carlsbad, Calif.). Integration results in gain of hygromycinresistance and loss of zeocin resistance and 1-galactosidase expression.The procedure is schematically shown in FIG. 9.

The attP or attB containing pcDNA/FRT plasmid DNAs were integrated intoFlp-In™-293 cells and clonal lines for each attP or attB site wereselected on media containing the hygromycin. As expected, these cellslost the β-galactosidase activity and were sensitive to zeocin. Thepresence of pcDNA/FRT plasmid with attP or attB sequence at the FRTlocus was also confirmed by PCR (FIG. 10). In PCR analysis, we detectedintegration of attP or attB sequence at the FRT locus in the genome byusing a primer that binds to attP or attB and another primer that bindsto adjacent FRT locus sequence. Therefore, the clone would be PCRpositive only if attP or attB site is integrated in the chromosome. Asexpected, the selected lines are positive for attP or attB. PCR did notamplify a specific band from the genomic DNA isolated from the parentalFlp-In™-293 cells (lanes P, Panel C in FIG. 10) but amplified a bandfrom the DNA isolated from cells integrated with attP or attB containingpcDNA/FRT plasmid (lanes 1, panel C in FIG. 10) for each recombinasetested. The stable 293 cells with attP or attB sites were used forintegrating plasmid containing the attB or attP sites, respectively.

Integration of Plasmid DNA at Chromosomal attP or attB Site

The integration assay plasmids were constructed by placing attP or attBsequence of each recombinase immediately before the puromycin resistancegene. In this plasmid, the puromycin gene does not have its ownpromoter. However, recombination between the attP on the chromosome andattB in the integration assay plasmid (or attB on the chromosome andattP on the assay plasmid) would integrate the puromycin gene next tothe CMV promoter present immediately before the attP or attB site in theFlp-In™-293 cells generated above (FIG. 9). The integration will resultin expression of puromycin gene and growth of such cells in the presenceof puromycin antibiotic. Random integration of assay plasmid is notexpected to provide resistance to puromycin. The Flp-In™-293 stable cellline containing the attP sequence was transfected with integration assayplasmid containing the attB site and with or without the correspondingrecombinase expression plasmid using the standard protocols. In anotherinstance, Flp-In™-293 stable cell line with stably integrated attBsequence were generated and used for integrating the attP containingintegration assay plasmid. Flp-In™-293 cells containing chromosomal attPor attB site (150,000 to 300,000 cells) were transfected with 100 ngintegration assay plasmid and 400 ng of recombinase expression plasmid.Cells were then selected on medium containing the puromycin antibiotic.If the recombinase is functional, the attB sequence containing plasmidis expected to integrate at the attP site on the chromosome or viceversa.

The number of puromycin resistant colonies obtained from attB or attPsite containing Flp-In™-293 cells after co-transfection with attP- orattB-containing integration assay plasmid and the correspondingrecombinase expression plasmid in 3 independent experiments is shown inTables 1 and 2 below. In the absence of recombinase plasmid, nopuromycin resistant colonies were observed. These results clearly showedthat the recombinases facilitated recombination between chromosomal attPor attB site and plasmid attB or attP site, resulting in integration ofplasmid DNA into chromosome. We also confirmed the plasmid integrationby isolating genomic DNA from puromycin resistant clones and detectedthe presence of attL and attR sites on the chromosome. Recombinationbetween attB and attP results in creation of attL and attR sites, whichare hybrid sites between attB and attP. PCR amplification using the attLor attR specific primers amplified the expected specific band only inpuromycin resistant clones after the integration of assay plasmid (lanesI, panels A and B in FIG. 10) but not in parental cells containing attPor attB that were used for integration (lanes P, panels A and B in FIG.10).

TABLE 1 Integration of attP containing plasmid into chromosome with attBsite Site Chromosomal on assay Number of puromycin^(R) clonesRecombinase site plasmid Exp #1 Exp #2 Exp #3 A118 attB attP 28 12 0SF370.1 attB attP Not done 48 148 SPβc2 attB attP 77 303 270 φRvl attBattP 4 9 0 Bxb1 attB attP 4 3 12

TABLE 2 Integration of attB containing plasmid into chromosome with attPsite Site Chromosomal on assay Number of puromycin^(R) clonesRecombinase site plasmid Exp #1 Exp#2 Exp #3 118 attP attB 34 55 26SF370.1 attP attB 0 2 2 SPβc2 attP attB 268 293 445 Bxbl attP attB 12 8Not done

Example 4 Deletion of Chromosomal DNA Flanked by attP and attB

Assay for the deletion of attP:STOP:attB sequence located on thechromosome was done in a two-step process. In the first step, stablecell lines containing a single copy of CMVpromoter—attP:STOP:attB—Luciferase gene—Terminator construct weregenerated for each recombinase and characterized. In the second step,recombinase expression plasmid was transiently transfected into stablecells with CMV promoter—attP:STOP:attB—Luciferase gene—Terminator andthe cells were assayed for the luciferase activity. If the recombinaseis active in mammalian cells, the recombination between chromosomal attPand attB sites will result in the deletion of STOP sequence andactivation of luciferase expression. The assay format is graphicallydepicted in FIG. 11.

Generation of Stable HEK293 Clones with CMVPromoter-attP-STOP-attB-Luciferase Gene Construct in the Chromosome

A single copy of CMV promoter—attP:STOP:attB—Luciferase gene—Terminatorconstruct was introduced at the FRT locus of Flp-In™-293 cells obtainedfrom Invitrogen, Carlsbad, Calif. (catalog #R750-07) as described above.The attP:STOP:attB—Luciferase gene sequence of each recombinase that waspresent in transient intramolecular recombination assay plasmids (seeDesign and construction of intramolecular recombination assay plasmidsand FIG. 1) was cloned into pcDNA/FRT plasmid (Invitrogen, Carlsbad,Calif., catalog #V6010-20) at the multiple cloning sites region presentbetween the between CMV promoter and BGH terminator sequence. Theconstructed pcDNA/FRT plasmid with CMVpromoter—attP:STOP:attB—Luciferase gene—Terminator was inserted at theFRT locus of Flp-In™-293 cells using Flp recombinase. Integration ofthis plasmid results in gain of hygromycin resistance and loss of zeocinresistance and β-galactosidase expression.

Flp-In™-293 cells were transfected with pcDNA/FRT plasmid containing theCMV promoter—attP:STOP:attB—Luciferase gene—Terminator along with Flpexpression plasmid (pCG44, Invitrogen, Carlsbad, Calif.). Clonesresistant to hygromycin were selected and expanded (FIG. 11). Theinsertion of pCDNA/FRT plasmid was also confirmed by assaying theselected clones for β-galactosidase activity. The selected clones lostthe β-galactosidase activity. The isolated clones were used fortransfection with recombinase expression plasmids.

Deletion of STOP Sequence from the Chromosome and Activation ofLuciferase in Stable Cell Lines

In the second step, hygromycin resistant cells containing the CMVpromoter—attP:STOP:attB—Luciferase gene—Terminator construct for eachrecombinase were transiently transfected with the correspondingrecombinase expression plasmid. Cells (15000 per well, 96-well format)were transfected with 0, 25, 50, 100, or 200 ng of recombinaseexpression plasmids and incubated for 24 hours. Cells were lysed with 50μl of passive lysis buffer and 25 μl extracts were assayed. Sixteenreplicate assays were performed, and luciferase activity (mean ofrelative light unit)±SE were plotted.

As shown in FIG. 12, transfection of increased amounts (0, 25, 50, 100,or 200 ng) of each recombinase expression plasmid into its correspondingattP:STOP:attB containing Flp-In™-293 clone increased the luciferaseactivity. These results showed that the recombinases can recombinechromosomally placed attP and attB sequences. The recombination resultedin the deletion of sequence flanked by attP and attB sites andactivation of luciferase gene.

Example 5 Integration of DNA at Chromosomal Pseudo Attachment Sites inHEK293 Cells

Assay for the insertion or integration of a plasmid containing attP orattB recombination site at the native pseudo attB or pseudo attP sitepresent in the HEK293 cell was done by co-transfecting cells with therecombinase expression plasmid and corresponding targeting plasmidcontaining the attP or attB site and hygromycin resistance gene, andselecting stable cells on media containing hygromycin antibiotic. Theprocedure is schematically depicted in FIG. 13. HEK293 cells weremaintained at 37′C and 5% CO₂ in DMEM supplemented with 10% fetal bovineserum and 1% penicillin/streptomycin (obtained from Invitrogen,Carlsbad, Calif.). On the day of transfection, cells were plated at adensity of 750,000 cells per 35 mm Petri dish. The cells weretransfected with 50 ng of targeting plasmid containing attP or attB siteand a Ubiquitin C promoter-driven hygromycin resistance gene (FIG. 13)alone or along with 4 μg of recombinase expression plasmid usingLipofectamine 2000™ according to the manufacturers instructions(Invitrogen, Carlsbad, Calif.). The chromosomal integration of plasmidwill result in expression of hygromycin gene and growth of such cells inthe presence of hygromycin antibiotic. It should be noted that randomintegration of targeting plasmid (i.e., at non-pseudo sites) could alsoresult in generation of hygromycin resistant clones. However, when thetarget plasmid is introduced into cells along with the recombinaseexpression plasmid, the number of hygromycin resistant HEK293 clones isexpected to be higher if the genome contains pseudo attachment sites.Also, for instance, if the integration is due to recombination betweenpseudo attB site on the genome and attP site on the targeting plasmidthe attP site on the targeting plasmid is precisely cut and plasmid isinserted at the pseudo attB sites in the genome, resulting in creationof pseudo attL and pseudo attR sites that can be identified by DNAsequencing of rescued plasmids. In contrast, random integrationsgenerally preserve the intact attP site after integration.

The hygromycin resistant HEK293 clones obtained in the presence ofrecombinase expression plasmid were pooled, genomic DNA preparation wasmade and digested with restriction enzymes that cut out side theintegrated plasmid (i.e., outside the region of pUC ori and bacterialselectable marker gene), the digested DNA was self-ligated, and theligated DNA was transformed into E. coli to rescue the integratedplasmid containing the adjacent genomic DNA, following the procedurescommon in this field (Thyagarajan, B. et al. (2001) Site-specificgenomic integration in mammalian cells mediated by phage φC31 integrase.Mol. Cell. Biol. 21: 3926-3934). Genomic DNA prepared from hygromycinresistant clones (10 μg) was digested with restriction enzymes Bgl II,Xba I, Eco 0109I, Ban II, Sty 1, Bso BI, or Btg I in 40 μL total volumefor 3 hrs @37° C. 20 μL of each digestion was ligated in 200 μL totalvolume overnight at 4° C., and then purified. The ligated DNA wasintroduced into E. coli by electroporation and ampicillin-resistant E.coli colonies were then selected on a plate containing the antibiotic.Plasmid DNAs was prepared from the bacterial colonies and the rescuedplasmid DNAs were then sequenced. The recovered genomic DNA sequence wasused to identify its chromosomal location by aligning the recoveredgenomic sequence with the human genome sequence at Genbank, NIH Libraryof Medicine using the BLAST program (http://www.ncbi.nlm.nih.gov/BLAST).

When the pseudo site targeting plasmid containing the attP site ofSF370.1 or SPβc2 recombinase was introduced into HEK293 cells, 9 and 0hygromycin resistant clones were obtained, respectively (Table 3). Incontrast, when the targeting plasmid DNA was co-introduced into HEK293cells along with respective SF370.1 or SPβc2 recombinase expressionplasmid, more than 100 hygromycin resistant clones were recovered ineach case (Table 3). These results clearly indicate thatrecombinase-mediated integration at chromosomal pseudo attB sites washighly efficient and integration at pseudo sites was many fold higherthan random integration of targeting plasmid (i.e., integration in theabsence of recombinase). Genomic DNA was isolated from pooledhygromycin-resistant HEK293 clones obtained with SF370.1 recombinase,plasmids were rescued from the genome, and pseudo attB sequences wereidentified by sequencing 100 plasmid DNAs as described above. Out of the100 rescued plasmids sequenced; there were 41 different pseudo attBsites, as there were more integrations at some pseudo sites than atother pseudo sites. For example, 35 out 100 recovered integrations wereat a single site. The nucleotide sequence of this pseudo attB site isgiven in FIG. 14. These results suggest that the SF370.1 recombinasepreferentially integrated plasmid DNA at this site compared to othersites.

TABLE 3 Integration of attP containing plasmid into HEK293 chromosomalpseudo attB sites Number of puromycin^(R) clones Recom- Chromosomal Siteon targeting Without Without binase pseudo site plasmid RecombinaseRecombinase SF370.1 attB attP 9 >100 SPβc2 attB attP 0 >100

Similar analysis was done with hygromycin resistant HEK293 clonesobtained after targeting of SPβc2 attP containing plasmid using theSPβc2 recombinase and 109 rescued plasmids DNAs were sequenced. Sequenceanalysis showed that 105 out of 107 integrations were at pseudo attBsites and 2 integrations were at random sites. There were 54 differentpseudo attB integration sites among the 105 integration sites recovered.Fifteen of the integrations occurred at one pseudo site sequence shownin FIG. 14. These results show that human and eukaryotic chromosomesserve as efficient targets for precise site-specific integrations atpseudo at sites using the enzymes we discovered. These sites formnaturally occurring targets for integration that can be used in manybiotechnology and medical applications.

1. A method for obtaining site-specific recombination in a eukaryoticcell, the method comprising: providing a eukaryotic cell that comprisesa first recombination site and a second recombination site: a stopsequence positioned between said first recombination site and saidsecond recombination site; contacting the first and second recombinationsites with a prokaryotic recombinase polypeptide, resulting inrecombination between the recombination sites and deletion of said stopsequence, wherein the recombinase polypeptide can mediate recombinationbetween the first and second recombination sites, the firstrecombination site is a phage genomic recombination attachment site(attP) or a bacterial genomic recombination attachment site (attB), thesecond recombination site is attB or attP, and the recombinase is aMycobacterium smegmatis Bxb1 phage recombinase, provided that when thefirst recombination attachment site is attB, the second recombinationattachment site is attP, and when the first recombination attachmentsite is attP, the second recombination attachment site is attB.
 2. Amethod for obtaining site-specific recombination in a eukaryotic cell,the method comprising: providing a eukaryotic cell that comprises afirst recombination site and a second recombination site; contacting thefirst and second recombination sites with a prokaryotic recombinasepolypeptide, resulting in recombination between the recombination sites,wherein the recombinase polypeptide can mediate recombination betweenthe first and second recombination sites, the first recombination siteis attP or attB, the second recombination site is a pseudo attachmentsite, and the recombinase is Mycobacterium smegmatis Bxb1 phagerecombinase.
 3. (canceled)
 4. The method of claim 1, wherein the serinerecombinase-encoding polynucleotide is operably linked to a promoterwhich mediates expression of the polynucleotide in the eukaryotic cell.5. The method of claim 1, wherein the serine recombinase polypeptide isintroduced into the eukaryotic cell by expression of a polynucleotidethat encodes the serine recombinase polypeptide.
 6. The method of claim1, wherein the serine recombinase polypeptide is introduced into theeukaryotic cell as a polypeptide.
 7. The method of claim 1, wherein theserine recombinase polypeptide is introduced into the eukaryotic cell bymessenger RNA that encodes the serine recombinase polypeptide.
 8. Themethod of claim 1, wherein the site-specific recombination results inintegration, deletion, inversion, translocation or exchange of DNA.
 9. Amethod for obtaining a eukaryotic cell having a stably integratedpolynucleotide sequence, the method comprising: introducing apolynucleotide into a eukaryotic cell that comprises a firstrecombination attB or attP site, wherein the polynucleotide comprises aplurality of nucleic acid sequences of interest and a secondrecombination attP or attB site, and contacting the first and the secondrecombination sites with a prokaryotic recombinase polypeptide, whereinthe recombinase polypeptide can mediate site specific recombinationbetween the first and second recombination sites, and the recombinase isa Mycobacterium smegmatis Bxb1 phage recombinase, provided that when thefirst recombination site is attB, the second recombination site is attPand when the first recombination site is attP, the second recombinationsite is attB.
 10. A method for obtaining a eukaryotic cell having astably integrated polynucleotide sequence, the method comprising:introducing a polynucleotide into a eukaryotic cell that comprises afirst recombination pseudo attachment site, wherein the polynucleotidecomprises a nucleic acid sequence and a second recombination attP orattB site, and contacting the first and the second recombination siteswith a prokaryotic recombinase polypeptide, wherein the recombinasepolypeptide can mediate site-specific recombination between the firstand second recombination sites, and the recombinase is a Mycobacteriumsmegmatis Bxb1 phage recombinase.
 11. (canceled)
 12. The method of claim9, wherein the serine recombinase-encoding polynucleotide is operablylinked to a promoter which mediates expression of the polynucleotide inthe eukaryotic cell.
 13. The method of claim 9, wherein the serinerecombinase polypeptide is introduced into the eukaryotic cell byexpression of a polynucleotide that encodes the serine recombinasepolypeptide.
 14. The method of claim 9, wherein the serine recombinasepolypeptide is introduced into the eukaryotic cell as a polypeptide. 15.The method of claim 9, wherein the serine recombinase polypeptide isintroduced into the eukaryotic cell by expression of RNA that encodesthe serine recombinase polypeptide.
 16. A method for obtainingsite-specific recombination in a eukaryotic cell, the method comprising:providing a eukaryotic cell that comprises a first recombination siteand a second recombination site with a polynucleotide sequence flankedby a third recombination site and a fourth recombination site;contacting the recombination sites with a prokaryotic recombinasepolypeptide, resulting in recombination between the recombination sites,wherein the recombinase polypeptide can mediate recombination betweenthe first and third recombination sites and the second and fourthrecombination sites, the first and second recombination sites are attPor attB, the third and fourth recombination sites are attB or attP, andthe recombinase is a Mycobacterium smegmatis Bxb1 phage recombinase,provided that when the first and second recombination attachment sitesare attB, the third and fourth recombination attachment sites are attP,and when the first and second recombination attachment sites are attP,the third and fourth recombination attachment sites are attB. 17.(canceled)
 18. The method of claim 16, wherein the serine recombinasepolypeptide is introduced into the eukaryotic cell by expression of apolynucleotide that encodes the serine recombinase polypeptide.
 19. Themethod of claim 16, wherein the serine recombinase polypeptide isintroduced into the eukaryotic cell as a polypeptide.
 20. The method ofclaim 16, wherein the serine recombinase polypeptide is introduced intothe eukaryotic cell by messenger RNA that encodes the serine recombinasepolypeptide. 21-28. (canceled)
 29. A method for the site-specificintegration of a polynucleotide of interest into the genome of atransgenic subject, wherein said genome comprises a first recombinationattB or attP site or pseudo attB or pseudo attP site, the methodcomprising: introducing a nucleic acid that comprises the polynucleotideof interest and a second recombination attP or attB site; contacting thefirst and the second recombination sites with a prokaryotic recombinasepolypeptide, wherein the recombinase polypeptide can mediatesite-specific recombination between the first and second recombinationsites, and the recombinase is a Mycobacterium smegmatis Bxb1 phagerecombinase, provided that when the first recombination site is attB orpseudo attB, the second recombination site is attP and when the firstrecombination site is attP or pseudo attP, the second recombination siteis attB. 30-35. (canceled)
 36. The method of claim 2, wherein the serinerecombinase-encoding polynucleotide is operably linked to a promoterwhich mediates expression of the polynucleotide in the eukaryotic cell.37. The method of claim 2, wherein the serine recombinase polypeptide isintroduced into the eukaryotic cell by expression of a polynucleotidethat encodes the serine recombinase polypeptide.
 38. The method of claim2, wherein the serine recombinase polypeptide is introduced into theeukaryotic cell as a polypeptide.
 39. The method of claim 2, wherein theserine recombinase polypeptide is introduced into the eukaryotic cell bymessenger RNA that encodes the serine recombinase polypeptide.
 40. Themethod of claim 2, wherein the site-specific recombination results inintegration, deletion, inversion, translocation or exchange of DNA. 41.The method of claim 10, wherein the serine recombinase-encodingpolynucleotide is operably linked to a promoter which mediatesexpression of the polynucleotide in the eukaryotic cell.
 42. The methodof claim 10, wherein the serine recombinase polypeptide is introducedinto the eukaryotic cell by expression of a polynucleotide that encodesthe serine recombinase polypeptide.
 43. The method of claim 10, whereinthe serine recombinase polypeptide is introduced into the eukaryoticcell as a polypeptide.
 44. The method of claim 10, wherein the serinerecombinase polypeptide is introduced into the eukaryotlc cell byexpression of RNA that encodes the serine recombinase polypeptide.